Magnetic resonance imaging agents for the detection of physiological agents

ABSTRACT

The invention relates to novel magnetic resonance imaging contrast agents and methods of detecting physiological signals or substances.

This application is a continuing application of U.S. Ser. No.08/460,511, filed Jun. 2, 1995 now abandoned; Ser. No. 08/486,968, filedJun. 7, 1995, now U.S. Pat. No. 5,707,605; Provisional Ser. No.60/063,328, filed Oct. 27, 1997; and Ser. No. 08/971,855, filed as PCTUS96/08549 Jun. 3, 1996 now abandoned.

FIELD OF THE INVENTION

The invention relates to novel magnetic resonance imaging contrastagents and methods of detecting physiological signals or substances.

BACKGROUND OF THE INVENTION

Magnetic resonance imaging (MRI) is a diagnostic and research procedurethat uses high magnetic fields and radio-frequency signals to produceimages. The most abundant molecular species in biological tissues iswater. It is the quantum mechanical "spin" of the water proton nucleithat ultimately gives rise to the signal in all imaging experiments. InMRI the sample to be imaged is placed in a strong static magnetic field(1-12 Tesla) and the spins are excited with a pulse of radio frequency(RF) radiation to produce a net magnetization in the sample. Variousmagnetic field gradients and other RF pulses then act on the spins tocode spatial information into the recorded signals. MRI is able togenerate structural information in three dimensions in relatively shorttime spans.

The Image.

MR images are typically displayed on a gray scale with black the lowestand white the highest measured intensity (I). This measured intensityI=C*M, where C is the concentration of spins (in this case, waterconcentration) and M is a measure of the magnetization present at timeof the measurement. Although variations in water concentration (C) cangive rise to contrast in MR images, it is the strong dependence of therate of change of M on local environment that is the source of imageintensity variation in MRI. Two characteristic relaxation times, T₁ &T₂, govern the rate at which the magnetization can be accuratelymeasured. T₁ is the exponential time constant for the spins to decayback to equilibrium after being perturbed by the RF pulse. In order toincrease the signal-to-noise ratio (SNR) a typical MR imaging scan (RF &gradient pulse sequence and data acquisition) is repeated at a constantrate for a predetermined number of times and the data averaged. Thesignal amplitude recorded for any given scan is proportional to thenumber of spins that have decayed back to equilibrium since the previousscan. Thus, regions with rapidly decaying spins (i.e. short T₁ values)will recover all of their signal amplitude between successive scans.

The measured intensities in the final image will accurately reflect thespin density (i.e. water content). Regions with long T₁ values comparedto the time between scans will progressively lose signal until a steadystate condition is reached and will appear as darker regions in thefinal image. Changes in T₂ (spin-spin relaxation time) result in changesin the signal linewidth (shorter T₂ values) yielding larger linewidths.In extreme situations the linewidth can be so large that the signal isindistinguishable from background noise. In clinical imaging, waterrelaxation characteristics vary from tissue to tissue, providing thecontrast which allows the discrimination of tissue types. Moreover, theMRI experiment can be setup so that regions of the sample with short T₁values and/or long T₂ values are preferentially enhanced so called T₁-weighted and T₂ -weighted imaging protocol.

MRI Contrast Agents.

There is a rapidly growing body of literature demonstrating the clinicaleffectiveness of paramagnetic contrast agents (currently 8 are inclinical trials or in use). The capacity to differentiateregions/tissues that may be magnetically similar but histologicallydistinct is a major impetus for the preparation of these agents [1, 2].In the design of MRI agents, strict attention must be given to a varietyof properties that will ultimately effect the physiological outcomeapart from the ability to provide contrast enhancement [3]. Twofundamental properties that must be considered are biocompatability andproton relaxation enhancement. Biocompatability is influenced by severalfactors including toxicity, stability (thermodynamic and kinetic),pharmacokinetics and biodistribution. Proton relaxation enhancement (orrelaxivity) is chiefly governed by the choice of metal and rotationalcorrelation times.

The first feature to be considered during the design stage is theselection of the metal atom, which will dominate the measured relaxivityof the complex. Paramagnetic metal ions, as a result of their unpairedelectrons, act as potent relaxation enhancement agents. They decreasethe T₁ and T₂ relaxation times of nearby (r⁶ dependence) spins. Someparamagnetic ions decrease the T₁ without causing substantiallinebroadening (e.g. gadolinium (III), (Gd³⁺)), while others inducedrastic linebroadening (e.g. superparamagnetic iron oxide). Themechanism of T₁ relaxation is generally a through space dipole-dipoleinteraction between the unpaired electrons of the paramagnet (the metalatom with an unpaired electron) and bulk water molecules (watermolecules that are not "bound" to the metal atom) that are in fastexchange with water molecules in the metal's inner coordination sphere(are bound to the metal atom).

For example, regions associated with a Gd³⁺ ion (near-by watermolecules) appear bright in an MR image where the normal aqueoussolution appears as dark background if the time between successive scansin the experiment is short (i.e. T₁ weighted image). Localized T₂shortening caused by superparamagnetic particles is believed to be dueto the local magnetic field inhomogeneities associated with the largemagnetic moments of these particles. Regions associated with asuperparamagnetic iron oxide particle appear dark in an MR image wherethe normal aqueous solution appears as high intensity background if theecho time (TE) in the spin-echo pulse sequence experiment is long (i.e.T₂ -weighted image). The lanthanide atom Gd³⁺ is by the far the mostfrequently chosen metal atom for MRI contrast agents because it has avery high magnetic moment (u² =63 BM²), and a symmetric electronicground state, (S⁸). Transition metals such as high spin Mn(II) andFe(III) are also candidates due to their high magnetic moments.

Once the appropriate metal has been selected, a suitable ligand orchelate must be found to render the complex nontoxic. The term chelatoris derived from the Greek word chele which means a "crabs claw", anappropriate description for a material that uses its many "arms" to graband hold on to a metal atom (see DTPA below). Several factors influencethe stability of chelate complexes include enthalpy and entropy effects(e.g. number, charge and basicity of coordinating groups, ligand fieldand conformational effects). Various molecular design features of theligand can be directly correlated with physiological results. Forexample, the presence of a single methyl group on a given ligandstructure can have a pronounced effect on clearance rate. While theaddition of a bromine group can force a given complex from a purelyextracellular role to an effective agent that collects in hepatocytes.

Diethylenetriaminepentaacetic (DTPA) chelates and thus acts to detoxifylanthanide ions. The stability constant (K) for Gd(DTPA)²⁻ is very high(logK=22.4) and is more commonly known as the formation constant (thehigher the logK, the more stable the complex). This thermodynamicparameter indicates the fraction of Gd³⁺ ions that are in the unboundstate will be quite small and should not be confused with the rate(kinetic stability) at which the loss of metal occurs (k_(f) /k_(d)).The water soluble Gd(DTPA)²⁻ chelate is stable, nontoxic, and one of themost widely used contrast enhancement agents in experimental andclinical imaging research. It was approved for clinical use in adultpatients in June of 1988. It is an extracellular agent that accumulatesin tissue by perfusion dominated processes.

To date, a number of chelators have been used, includingdiethylenetriaminepentaacetic (DTPA),1,4,7,10-tetraazacyclododecane'-N,N'N",N'"-tetracetic acid (DOTA), andderivatives thereof. See U.S. Pat. Nos. 5,155,215, 5,087,440, 5,219,553,5,188,816, 4,885,363, 5,358,704, 5,262,532, and Meyer et al., Invest.Radiol. 25: S53 (1990).

Image enhancement improvements using Gd(DTPA) are well documented in anumber of applications (Runge et al., Magn, Reson. Imag. 3:85 (1991);Russell et al., AJR 152:813 (1989); Meyer et al., Invest. Radiol. 25:S53(1990)) including visualizing blood-brain barrier disruptions caused byspace occupying lesions and detection of abnormal vascularity. It hasrecently been applied to the functional mapping of the human visualcortex by defining regional cerebral hemodynamics (Belliveau et al.,(1991) 254:719).

Another chelator used in Gd contrast agents is the macrocyclic ligand1,4,7,10-tetraazacyclododecane-N,N',N"N'"-tetracetic acid (DOTA). TheGd-DOTA complex has been thoroughly studied in laboratory testsinvolving animals and humans. The complex is conformationally rigid, hasan extremely high formation constant (logK=28.5), and at physiologicalpH possess very slow dissociation kinetics. Recently, the GdDOTA complexwas approved as an MRI contrast agent for use in adults and infants inFrance and has been administered to over 4500 patients.

As noted above, these MRI contrast agents have a variety of uses.However, there are no MRI contrast agents that report on physiologic ormetabolic processes within a biological or other type of sample.Accordingly, it is an object of the present invention to provide MRIcontrast or enhancement agents which allow the visualization anddetection of physiological agents within an animal, tissue or cells.

SUMMARY OF THE INVENTION

In accordance with the above objects, the invention provides MRI agentscomprising a paramagnetic metal ion bound to a complex. The complexcomprises a chelator and a blocking moiety in at least a firstcoordination sites of said metal ion. The blocking moiety is covalentlyattached to the chelator, and capable of interacting with a targetsubstance such that the exchange of water in at least said firstcoordination site in the metal ion complex is altered.

In one aspect, the invention provides MRI agents comprising a) a Gd(III)ion bound to a chelator such that the Gd(III) ion has coordination atomsin at least 5 coordination sites, and b) a blocking moiety covalentlyattached to the chelator which hinders the rapid exchange of water inthe remaining coordination sites. The blocking moiety is capable ofinteracting with a target substance such that the exchange of water inthe remaining coordination sites is increased.

In an additional aspect, the invention provides MRI agents having theformula: ##STR1## wherein M is a paramagnetic metal ion selected fromthe group consisting of Gd(III), Fe(III), Mn(II), Yt(III), Cr(III) andDy(III);

A, B, C and D are either single bonds or double bonds;

X₁, X₂, X₃ and X₄ are --OH, --COO--, --CH₂ OH--CH₂ COO--, or a blockingmoiety;

R₁ -R₁₂ are hydrogen, alkyl, aryl, phosphorus moiety, or a blockingmoiety;

wherein at least one of X₁ -X₄ and R₁ -R₁₂ is a blocking moiety.

In a further aspect, the invention provides MRI contrast agentscomprising a first paramagnetic metal ion bound to a first complex, andat least a second paramagnetic metal ion bound to a second complex. Thefirst and second complexes each comprise a chelator with a covalentlyattached blocking moiety. The complexes can be attached via a linker,for example a polymer.

In an additional aspect, the MRI agent comprise a) a first chelatorcomprising a first paramagnetic metal ion; b) a second chelatorcomprising a second paramagnetic metal ion; and c) a blocking moietycovalently attached to at least one of the first or second chelators.The blocking moiety provides at least a first coordination atom of eachof the first and second metal ions, or serves as a coordination sitebarrier. As above, the blocking moiety is capable of interacting with atarget substance such that the exchange of water in at least a firstcoordination site of at least one of the metal ions is increased.

The invention also provides methods of magnetic resonance imaging of acell, tissue, experimental animal or patient comprising administering anMRI agent of the invention to a cell, tissue, experimental animal orpatient and rendering a magnetic resonance image of said cell, tissue,experimental animal or patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a representative complex of the invention, where theblocking moiety is tethered at one end only. The blocking moietycomprises a enzyme substrate and a coordination site barrier. The Rgroup is the coordination site barrier.

FIG. 2 depicts a representative complex of the invention, wherein theblocking moiety is tethered at two ends. The R group is the coordinationsite barrier.

FIG. 3 depicts a representative synthesis ofDo3a-hydroxyethyl-β-galactose, which has a single galactose moietyattached to the DOTA ring.

FIG. 4 depicts a representative synthesis of a β-galactose-DOTAderivative that has two galactose moieties attached to the DOTA ring.

FIG. 5 depicts the synthesis of a BAPTA-DOTA derivative.

FIG. 6 depicts the syntheis of a FURA-DOTA derivative.

FIG. 7 depicts a synthetic scheme for the synthesis of BAPTA-DTPA.

FIG. 8 depicts an alternative synthesis of a BAPTA-DTPA derivative.

FIG. 9 depicts the change in T₁ observed upon the β-galactosidasecatalyzed cleavage of the galactopyranose residue (n=3). The firstColumn of each pair (1,3,5) represents the T₁ of the galactose-DOTAcomplex and β-galactosidase mixture immediately after addition. Thesecond column represents the T₁ of the solution after a period of timein the presence of β-galactosidase. Each column is reported as a ratioto a control containing only the complex. Column 1 and 2: 2.0 mM Gdcomplex plus 1.7 uM β-galactosidase phosphate buffer (25 mM) pH 7.3.Column 3 and 4: 2.0 mM Gd plus 5.1 uM β-galactosidase phosphate buffer(25 mM) pH 7.3. Column 5 and 6: 2 mM Gd complex plus 5.1 uM heatinactivated β-galactosidase (10 minutes at 80 degrees) phosphate buffer(25 mM) pH 7.3. The complexes were incubated with the enzyme for 7 daysand HPLC traces indicated greater than 95% cleavage. A minimalconcentration of enzyme was used in these experiments to reducepotential effects of any contrast agent-enzyme interactions. T₁ werecarried out using a Bruker AMX 500 spectrometer at 26 degrees using astandard inversion-recovery sequence. The solution was placed in a 40 ulround bottomed NMR tube insert (Wilmad glass) and inserted into a tubecontaining d₃ -chloroform. A two dimensional data file was collectedcontaining 16 different inversion delays with 8 scans each. The raw nmrdata was processed (Felix, BIOSYM/Molecular Simulations, San Diego,Calif.) and the peak heights were fitted to an exponential rise to a maxto obtain T₁. The R value was always greater than 0.999.

FIGS. 10A, 10B, 10C, 10D, 10E, 10F, and 10G depict several of thepossible conformations of the dimer embodiments. Boxes representchelators, with M being the paramagnetic metal ions. FIGS. 10A and 10Brepresent two possible duplex conformations. In FIG. 10A, R₂₇ can be alinker, such as described herein as R₂₆, a cleavable moiety such as anenzyme substrate such as a peptide, or a blocking moiety that willpreferentially interact with the target molecule. R₂₈, which may or maynot be present depending on R₂₇, is a coordination site barrier similarto R₂₃ or a blocking moiety. FIG. 10B has R₂₈ blocking moieties orcoordination site barriers attached via an R₂₇ group to two chelators.FIG. 10C is similar to FIG. 10A, but at least one of the R₂₇ groups mustbe a cleavable moiety. FIG. 10D depicts the case where two blockingmoieties or coordination site barriers are present; if R₂₇ is a blockingmoiety, R₂₈ need not be present. FIG. 10E is similar to 10B but thechelators need not be covalently attached. FIGS. 10F (single MRI agents)and and 10G (duplex agents) are multimers of MRI contrast agents,wherein n can be from 1 to 1000, with from 1 to about 20 beingpreferred, and from about 1 to 10 being especially preferred.

FIGS. 10H and 10I depict polymer 10 as defined herein being attached toeither single MRI agents (10H) or duplex MRI agents (10I).

FIGS. 11A, 11B and 11C depicts precursors for making MRI duplexes forCa⁺² detection using BAPTA derivatives as the blocking moiety, each witha different R₂₆ linkers. FIG. 11A depicts AEPA, which when Gd is presentexhibits a q of 0.7 (q is the number of water molecules associated withthe complex, which is an indicator of the ability of the blocking moietyto block the exchange of water; the lower the q the better). The qvalues were determined using fluorescence lifetime measurements usingTerbium (Tb³⁺) as the metal ion in D₂ O and H₂ O (data not shown). FIG.11B depicts APPA, which has a q of 0.3. FIG. 11C depicts ABPA, which hasa q of 0.7.

FIG. 12 depicts the synthesis of AEPA. As will be appreciated by thosein the art, the full duplex can be made by functionalizing the otherortho position on the nitrobenzyl ring.

FIG. 13 depicts the synthesis of APPA and ABPA. As will be appreciatedby those in the art, the full duplexes can be made by functionalizingthe other ortho position on the nitrobenzyl ring.

FIG. 14 depicts the synthesis of Gd3+-BAPTA-DO3A₂ ("CalGad").

FIG. 15 schematically depicts the structural changes in CalGad thatoccur upon binding of calcium.

FIG. 16 depicts the relaxivity of the CalGad complex as a function ofcalcium ion concentration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides magnetic resonance imaging contrastagents which can detect physiological agents or target substances. TheMRI agents of the invention are relatively inactive, or have weakrelaxivity, as contrast enhancement agents in the absence of thephysiological target substance, and are activated, thus altering the MRimage, in the presence of the physiological target substance.

Viewed simplistically, this "trigger" mechanism, whereby the contrastagent is "turned on" (i.e. increases the relaxivity) by the presence ofthe target substance, is based on a dynamic equilibrium that affects therate of exchange of water molecules in one or more coordination sites ofa paramagnetic metal ion contained in the MRI contrast agents of thepresent invention. In turn, the rate of exchange of the water moleculeis determined by the presence or absence of the target substance in thesurrounding environment. Thus, in the absence of the target substance,the metal ion complexes of the invention which chelate the paramagneticion have reduced coordination sites available which can rapidly exchangewith the water molecules of the local environment. In such a situation,the water coordination sites are substantially occupied or blocked bythe coordination atoms of the chelator and at least one blocking moiety.Thus, the paramagnetic ion has essentially no water molecules in its"inner-coordination sphere", i.e. actually bound to the metal when thetarget substance is absent. It is the interaction of the paramagneticmetal ion with the protons on the inner coordination sphere watermolecules and the rapid exchange of such water molecules that cause thehigh observed relaxivity, and thus the imaging effect, of theparamagnetic metal ion. Accordingly, if all the coordination sites ofthe metal ion in the metal ion complex are occupied with moieties otherthan water molecules, as is the case when the target substance isabsent, there is little if any net enhancement of the imaging signal bythe metal ion complexes of the invention. However, when present, thetarget substance interacts with the blocking moiety or moities of themetal ion complex, effectively freeing at least one of the inner-spherecoordination sites on the metal ion complex. The water molecules of thelocal environment are then available to occupy the inner-spherecoordination site or sites, which will cause an increase in the rate ofexchange of water and relaxivity of the metal ion complex toward waterthereby producing image enhancement which is a measure of the presenceof the target substance.

Generally, a 2 to 5% change in the MRI signal used to generate the imageis sufficient to be detectable. Thus, it is preferred that the agents ofthe invention in the presence of a target substance increase the MRIsignal by at least 2 to 5% as compared to the signal gain the absence ofthe target substance. Signal enhancement of 2 to 90% is preferred, and10 to 50% is more preferred for each coordination site made available bythe target substance interaction with the blocking moiety. That is, whenthe blocking moiety occupies two or more coordination sites, the releaseof the blocking moiety can result in double the increase in signal ormore as compared to a single coordination site.

It should be understood that even in the absence of the targetsubstance, at any particular coordination site, there will be a dynamicequilibrium for one or more coordination sites as between a coordinationatom of the blocking moiety and water molecules. That is, even when acoordination atom is tightly bound to the metal, there will be someexchange of water molecules at the site. However, in most instances,this exchange of water molecules is neither rapid nor significant, anddoes not result in significant image enhancement. However, upon exposureto the target substance, the blocking moiety dislodges from thecoordination site and the exchange of water is increased, i.e. rapidexchange and therefore an increase in relaxivity may occur, withsignificant image enhancement.

The complexes of the invention comprise a chelator and a blockingmoiety. The metal ion complexes of the invention comprise a paramagneticmetal ion bound to a complex comprising a chelator and a blockingmoiety. By "paramagnetic metal ion", "paramagnetic ion" or "metal ion"herein is meant a metal ion which is magnetized parallel or antiparallelto a magnetic field to an extent proportional to the field. Generally,these are metal ions which have unpaired electrons; this is a termunderstood in the art. Examples of suitable paramagnetic metal ions,include, but are not limited to, gadolinium III (Gd+3 or Gd(III)), ironIII (Fe+3 or Fe(III)), manganese II (Mn+2 or Mn(II)), yttrium III (Yt+3or Yt(III)), dysprosium (Dy+3 or Dy(III)), and chromium (Cr(III) orCr+3). In a preferred embodiment the paramagnetic ion is the lanthanideatom Gd(III), due to its high magnetic moment (u² =63 BM2), a symmetricelectronic ground state (S8), and its current approval for diagnosticuse in humans.

In addition to the metal ion, the metal ion complexes of the inventioncomprise a chelator and a blocking moiety which may be covalentlyattached to the chelator. Due to the relatively high toxicity of many ofthe paramagnetic ions, the ions are rendered nontoxic in physiologicalsystems by binding to a suitable chelator. Thus, the substitution ofblocking moieties in coordination sites of the chelator, which in thepresence of the target substance are capable of vacating thecoordination sites in favor of water molecules, may render the metal ioncomplex more toxic by decreasing the half-life of dissociation for themetal ion complex. Thus, in a preferred embodiment, only a singlecoordination site is occupied or blocked by a blocking moeity. However,for some applications, e.g. analysis of tissue and the like, thetoxicity of the metal ion complexes may not be of paramount importance.Similarly, some metal ion complexes are so stable that even thereplacement of one or more additional coordination atoms with a blockingmoiety does not significantly effect the half-life of dissociation. Forexample, DOTA, described below, when complexed with Gd(III) is extremelystable. Accordingly, when DOTA serves as the chelator, several of thecoordination atoms of the chelator may be replaced with blockingmoieties without a significant increase in toxicity. Additionally suchan agent would potentially produce a larger signal since it has two ormore coordination sites which are rapidly exchanging water with the bulksolvent.

There are a variety of factors which influence the choice and stabilityof the chelate metal ion complex, including enthalpy and entropy effects(e.g. number, charge and basicity of coordinating groups, ligand fieldand conformational effects).

In general, the chelator has a number of coordination sites containingcoordination atoms which bind the metal ion. The number of coordinationsites, and thus the structure of the chelator, depends on the metal ion.The chelators used in the metal ion complexes of the present inventionpreferably have at least one less coordination atom (n-1) than the metalion is capable of binding (n), since at least one coordination site ofthe metal ion complex is occupied or blocked by a blocking moeity, asdescribed below, to confer functionality on the metal ion complex. Thus,for example, Gd(III) may have 8 strongly associated coordination atomsor ligands and is capable of weakly binding a ninth ligand. Accordingly,suitable chelators for Gd(III) will have less than 9 coordination atoms.In a preferred embodiment, a Gd(III) chelator will have 8 coordinationatoms, with a blocking moiety either occupying or blocking the remainingsite in the metal ion complex. In an alternative embodiment, thechelators used in the metal ion complexes of the invention have two lesscoordination atoms (n-2) than the metal ion is capable of binding (n),with these coordination sites occupied by one or more blocking moieties.Thus, alternative embodiments utilize Gd(III) chelators with at least 5coordination atoms, with at least 6 coordination atoms being preferred,at least 7 being particularly preferred, and at least 8 being especiallypreferred, with the blocking moiety either occupying or blocking theremaining sites. It should be appreciated that the exact structure ofthe chelator and blocking moiety may be difficult to determine, and thusthe exact number of coordination atoms may be unclear. For example, itis possible that the chelator provide a fractional or non-integer numberof coordination atoms; i.e. the chelator may provide 7.5 coordinationatoms, i.e. the 8th coordination atom is on average not fully bound tothe metal ion. However, the metal ion complex may still be functional,if the 8th coordination atom is sufficiently bound to prevent the rapidexchange of water at the site, and/or the blocking moiety impedes therapid exchange of water at the site.

There are a large number of known macrocyclic chelators or ligands whichare used to chelate lanthanide and paramagnetic ions. See for example,Alexander, Chem. Rev. 95:273-342 (1995) and Jackels, Pharm. Med. Imag,Section III, Chap. 20, p645 (1990), expressly incorporated herein byreference, which describes a large number of macrocyclic chelators andtheir synthesis. Similarly, there are a number of patents which describesuitable chelators for use in the invention, including U.S. Pat. Nos.5,155,215, 5,087,440, 5,219,553, 5,188,816, 4,885,363, 5,358,704,5,262,532, and Meyer et al., Invest. Radiol. 25: S53 (1990), all ofwhich are also expressly incorportated by reference. Thus, as will beunderstood by those in the art, any of the known paramagnetic metal ionchelators or lanthanide chelators can be easily modified using theteachings herein to further comprise at least one blocking moiety.

When the metal ion is Gd(III), a preferred chelator is1,4,7,10-tetraazacyclododecane-N,N',N", N'"-tetracetic acid (DOTA) orsubstituted DOTA. DOTA has the structure shown below: ##STR2## By"substituted DOTA" herein is meant that the DOTA may be substituted atany of the following positions, as shown below: ##STR3## Suitable Rsubstitution groups include a wide variety of groups, as will beunderstood by those in the art. For example, suitable substitutiongroups include substitution groups disclosed for DOTA and DOTA-typecompounds in U.S. Pat. Nos. 5,262,532, 4,885,363, and 5,358,704. Thesegroups include hydrogen, alkyl groups including substituted alkyl groupsand heteroalkyl groups, aryl groups including substituted aryl andheteroaryl groups, phosphorus moieties, and blocking moieties. As willbe appreciated by those skilled in the art, each position designatedabove may have two R groups attached (R' and R"), although in apreferred embodiment only a single non-hydrogen R group is attached atany particular position; that is, preferably at least one of the Rgroups at each position is hydrogen. Thus, if R is an alkyl or arylgroup, there is generally an additional hydrogen attached to the carbon,although not depicted herein. In a preferred embodiment, one R group isa blocking moiety and the other R groups are hydrogen.

By "alkyl group" or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. Also included within the definitionof alkyl are heteroalkyl groups, wherein the heteroatom is selected fromnitrogen, oxygen, phosphorus, sulfur and silicon. Also included withinthe definition of an alkyl group are cycloalkyl groups such as C5 and C6rings, and heterocycloalkyl.

Additional suitable heterocyclic substituted rings are depicted in U.S.Pat. No. 5,087,440, expressly incorporated by reference. In someembodiments, two adjacent R groups may be bonded together to form ringstructures together with the carbon atoms of the chelator, such as isdescribed in U.S. Pat. No. 5,358,704, expressly incorporated byreference. These ring structures may be similarly substituted.

The alkyl group may range from about I to 20 carbon atoms (C1-C20), witha preferred embodiment utilizing from about 1 to about 10 carbon atoms(C1-C10), with about C1 through about C5 being preferred. However, insome embodiments, the alkyl group may be larger, for example when thealkyl group is the coordination site barrier.

By "alkyl amine" or grammatical equivalents herein is meant an alkylgroup as defined above, substituted with an amine group at any position.In addition, the alkyl amine may have other substitution groups, asoutlined above for alkyl group. The amine may be primary (--NH₂ R),secondary (--NHR₂), or tertiary (--NR₃). When the amine is a secondaryor tertiary amine, suitable R groups are alkyl groups as defined above.A preferred alkyl amine is p-aminobenzyl. When the alkyl amine serves asthe coordination site barrier, as described below, preferred embodimentsutilize the nitrogen atom of the amine as a coordination atom, forexample when the alkyl amine includes a pyridine or pyrrole ring.

By "aryl group" or grammatical equivalents herein is meant aromatic arylrings such as phenyl, heterocyclic aromatic rings such as pyridine,furan, thiophene, pyrrole, indole and purine, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus.

Included within the definition of "alkyl" and "aryl" are substitutedalkyl and aryl groups. That is, the alkyl and aryl groups may besubstituted, with one or more substitution groups. For example, a phenylgroup may be a substituted phenyl group. Suitable substitution groupsinclude, but are not limited to, halogens such as chlorine, bromine andfluorine, amines, hydroxy groups, carboxylic acids, nitro groups,carbonyl and other alkyl and aryl groups as defined herein. Thus,arylalkyl and hydroxyalkyl groups are also suitable for use in theinvention. Preferred substitution groups include alkyl amines and alkylhydroxy.

By "phosphorous moieties" herein is meant moieties containing the--PO(OH)(R₂₅)₂ group. The phosphorus may be an alkyl phosphorus; forexample, DOTEP utilizes ethylphosphorus as a substitution group on DOTA.R₂₅ may be alkyl, substituted alkyl, hydroxy. A preferred embodiment hasa --PO(OH)₂ R₂₅ group.

The substitution group may also be hydrogen or a blocking moiety, as isdescribed below.

In an alternative embodiment, when the metal ion is Gd(III), a preferredchelator is diethylenetriaminepentaacetic acid (DTPA) or substitutedDTPA. DPTA has the structure shown below: ##STR4##

By "substituted DPTA" herein is meant that the DPTA may be substitutedat any of the following positions, as shown below: ##STR5## See forexample U.S. Pat. No. 5,087,440.

Suitable R substitution groups include those outlined above for DOTA.Again, those skilled in the art will appreciate that there may be two Rgroups (R' and R") at each position designated above, although asdescribed herein, at least one of the groups at each position ishydrogen, which is generally not depicted herein.

In an alternative embodiment, when the metal ion is Gd(III), a preferredchelator is1,4,7,10-tetraazacyclododecane-N,N',N",N'"-tetraethylphosphorus (DOTEP)or substituted DOTEP (see U.S. Pat. No. 5,188,816). DOTEP has thestructure shown below: ##STR6## DOTEP may have similar R substitutiongroups as outlined above.

Other suitable Gd(III) chelators are described in Alexander, supra,Jackels, supra, U.S. Pat. Nos. 5,155,215, 5,087,440, 5,219,553,5,188,816, 4,885,363, 5,358,704, 5,262,532, and Meyer et al., Invest.Radiol. 25: S53 (1990), among others.

When the paramagnetic ion is Fe(III), appropriate chelators will haveless than 6 coordination atoms, since Fe(III) is capable of binding 6coordination atoms. Suitable chelators for Fe(III) ions are well knownin the art, see for example Lauffer et al., J. Am. Chem. Soc. 109:1622(1987); Lauffer, Chem. Rev. 87:901-927 (1987); and U.S. Pat. Nos.4,885,363, 5,358,704, and 5,262,532, all which describe chelatorssuitable for Fe(III).

When the paramagnetic ion is Mn(II) (Mn+2), appropriate chelators willhave less than 5 or 6 coordination atoms, since Mn(II) is capable ofbinding 6 or 7 coordination atoms. Suitable chelators for Mn(II) ionsare well known in the art; see for example Lauffer, Chem. Rev.87:901-927 (1987) and U.S. Pat. Nos. 4,885,363, 5,358,704, and5,262,532.

When the paramagnetic ion is Yt(III), appropriate chelators will haveless than 7 or 8 coordination atoms, since Yt(III) is capable of binding8 or 9 coordination atoms. Suitable chelators for Yt(III) ions include,but are not limited to, DOTA and DPTA and derivatives thereof (see Moiet al., J. Am. Chem. Soc. 110:6266-6267 (1988)) and those chelatorsdescribed in U.S. Pat. No. 4,885,363 and others, as outlined above.

When the paramagnetic ion is Dy+3 (Dy(III)), appropriate chelators willhave less than 7 or 8 coordination atoms, since DyIII is capable ofbinding 8 or 9 coordination atoms. Suitable chelators are known in theart, as above.

In a preferred embodiment, the chelator and the blocking moiety arecovalently linked; that is, the blocking moiety is a substitution groupon the chelator. In this embodiment, the substituted chelator, with thebound metal ion, comprises the metal ion complex which in the absence ofthe target substance has all possible coordination sites occupied orblocked; i.e. it is coordinatively saturated.

In an alternative embodiment, the chelator and the blocking moiety arenot covalently attached. In this embodiment, the blocking moiety hassufficient affinity for the metal ion to prevent the rapid exchange ofwater molecules in the absence of the target substance. However, in thisembodiment the blocking moiety has a higher affinity for the targetsubstance than for the metal ion. Accordingly, in the presence of thetarget substance, the blocking moiety will have a tendency to bedislodged from the metal ion to interact with the target substance, thusfreeing up a coordination site in the metal ion complex and allowing therapid exchange of water and an increase in relaxivity.

What is important is that the metal ion complex, comprising the metalion, the chelator and the blocking moiety, is not readily able torapidly exchange water molecules when the blocking moeities are in theinner coordination sphere of the metal ion, such that in the absence ofthe target substance, there is less or little substantial imageenhancement.

By "blocking moiety" or grammatical equivalents herein is meant afunctional group associated with the chelator metal ion complexes of theinvention which is capable of interacting with a target substance andwhich is capable, under certain circumstances, of substantially blockingthe exchange of water in at least one inner coordination site of themetal ion of the metal ion complex. For example, when bound to orassociated with the metal ion complexes of the invention, the blockingmoiety occupies or blocks at least one coordination site of the metalion in the absence of the target substance. Thus, the metal ion iscoordinately saturated with the chelator and the blocking moiety ormoieties in the absence of the target substance.

A blocking moiety may comprise several components. The blocking moietyhas a functional moiety which is capable of interacting with a targetsubstance, as outlined below. This functional moiety may or may notprovide the coordination atom(s) of the blocking moiety. In addition,blocking moieties may comprise one or more linker groups to allow forcorrect spacing and attachment of the components of the blocking moiety.Furthermore, in the embodiment where the functional group of theblocking moiety does not contribute a coordination atom, the blockingmoiety may comprise a coordination site barrier, which serves to eitherprovide a coordination site atom or sterically prevent the rapidexchange of water at the coordination site; i.e. the coordination sitebarrier may either occupy or block the coordination site.

By "capable of interacting with a target substance" herein is meant thatthe blocking moiety has an affinity for the target substance, such thatthe blocking moiety will stop blocking or occupying at least onecoordination site of the metal ion complex when the target substance ispresent. Thus, as outlined above, the blocking moiety is blocking oroccupying at least one coordination site of the metal ion in the absenceof the target substance. However, in the presence of the targetsubstance, the blocking moiety associates or interacts with the targetsubstance and is released from its association with the metal ion, thusfreeing at least one coordination site of the metal ion such that therapid exchange of water can occur at this site, resulting in imageenhancement.

The nature of the interaction between the blocking moiety and the targetsubstance will depend on the target substance to be detected orvisualized via MRI. For example, suitable target substances include, butare not limited to, enzymes; proteins; peptides; nucleic acids; ionssuch as Ca+2, Mg+2, Zn+2, K+, Cl-, and Na+; cAMP; receptors such ascell-surface receptors and ligands; hormones; antigens; antibodies; ATP;NADH; NADPH; FADH₂ ; FNNH₂ ; coenzyme A (acyl CoA and acetyl CoA); andbiotin, among others.

In some embodiments, the nature of the interaction is irreversible, suchthat the blocking moiety does not reassociate to block or occupy thecoordination site; for example, when the blocking moiety comprises anenzyme substrate which is cleaved upon exposure to the target enzyme.Alternatively, the nature of the interaction is reversible, such thatthe blocking moiety will reassociate with the complex to hinder theexchange of water; for example, when the blocking moiety comprises anion ligand, or a receptor ligand, as outlined below.

The corresponding blocking moieties will be enzyme substrates orinhibitors, receptor ligands, antibodies, antigens, ion bindingcompounds, substantially complementary nucleic acids, nucleic acidbinding proteins, etc.

In a preferred embodiment, the target substance is an enzyme, and theblocking moiety is an enzyme substrate. In this embodiment, the blockingmoiety is cleaved from the metal ion complex of the invention, allowingthe exchange of water in at least one coordination site of the metal ioncomplex. This embodiment allows the amplification of the imageenhancement since a single molecule of the target substance is able togenerate many activated metal ion complexes, i.e. metal ion complexes inwhich the blocking moiety is no longer occupying or blocking acoordination site of the metal ion.

As will be appreciated by those skilled in the art, the possible enzymetarget substances are quite broad. The target substance enzyme may bechosen on the basis of a correlation to a disease condition, forexample, for diagnositic purposes. Alternatively, the metal ioncomplexes of the present invention may be used to establish suchcorrelations.

Suitable classes of enzymes include, but are not limited to, hydrolasessuch as proteases, carbohydrases, lipases and nucleases; isomerases suchas racemases, epimerases, tautomerases, or mutases; transferases,kinases and phophatases.

As will be appreciated by those skilled in the art, the potential listof suitable enzyme targets is quite large. Enzymes associated with thegeneration or maintenance of arterioschlerotic plaques and lesionswithin the circulatory system, inflammation, wounds, immune response,tumors, may all be detected using the present invention. Enzymes such aslactase, maltase, sucrase or invertase, cellulase, α-amylase, aldolases,glycogen phosphorylase, kinases such as hexokinase, proteases such asserine, cysteine, aspartyl and metalloproteases may also be detected,including, but not limited to, trypsin, chymotrypsin, and othertherapeutically relevant serine proteases such as tPA and the otherproteases of the thrombolytic cascade; cysteine proteases including: thecathepsins, including cathepsin B, L, S, H, J, N and O; and calpain; andcaspases, such as caspase-3, -5, -8 and other caspases of the apoptoticpathway, and interleukin-converting enzyme (ICE). Similarly, bacterialand viral infections may be detected via characteristic bacterial andviral enzymes. As will be appreciated in the art, this list is not meantto be limiting.

Once the target enzyme is identified or chosen, enzyme substrateblocking moieties can be designed using well known parameters of enzymesubstrate specificities.

For example, when the enzyme target substance is a protease, theblocking moieity may be a peptide or polypeptide which is capable ofbeing cleaved by the target protease. By "peptide" or "polypeptide"herein is meant a compound of about 2 to about 15 amino acid residuescovalently linked by peptide bonds. Preferred embodiments utilizepolypeptides from about 2 to about 8 amino acids, with about 2 to about4 being the most preferred. Preferably, the amino acids are naturallyoccurring amino acids, although amino acid analogs and peptidomimiticstructures are also useful. Under certain circumstances, the peptide maybe only a single amino acid residue.

Similarly, when the enzyme target substance is a carbohydrase, theblocking moiety will be a carbohydrate group which is capable of beingcleaved by the target carbohydrase. For example, when the enzyme targetis lactase or β-galactosidase, the enzyme substrate blocking moiety islactose or galactose. Similar enzyme/blocking moiety pairs includesucrase/sucrose, maltase/maltose, and α-amylase/amylose.

In another embodiment, the blocking moiety may be an enzyme inhibitor,such that in the presence of the enzyme, the inhibitor blocking moietydisassociates from the metal ion complex to interact or bind to theenzyme, thus freeing an inner coordination sphere site of the metal ionfor interaction with water. As above, the enzyme inhibitors are chosenon the basis of the enzyme target substance and the corresponding knowncharacteristics of the enzyme.

In a preferred embodiment, the blocking moiety is a phosphorus moiety,as defined above, such as --(OPO(OR₂))_(n), wherein n is an integer from1 to about 10, with from 1 to 5 being preferred and 1 to 3 beingparticularly preferred. Each R is independently hydrogen or asubstitution group as defined herein, with hydrogen being preferred.This embodiment is particularly useful when the target molecule isalkaline phosphatase or a phosphodiesterase, or other enzymes known tocleave phosphorus containing moieties such as these.

In one embodiment, the blocking moiety is a nucleic acid. The nucleicacid may be single-stranded or double stranded, and includes nucleicacid analogs such as peptide nucleic acids and other well-knownmodifications of the ribose-phosphate backbone, such asphosphorthioates, phosphoramidates, morpholino structures, etc. Thetarget molecule can be a substantially complementary nucleic acid or anulceic acid binding moiety, such as a protein.

In a preferred embodiment, the target substance is a physiologicalagent. As for the enzyme/substrate embodiment, the physiological agentinteracts with the blocking moiety of the metal ion complex, such thatin the presence of the physiological agent, there is rapid exchange ofwater in at least one inner sphere coordination site of the metal ioncomplex. Thus, the target substance may be a physiologically active ion,and the blocking moiety is an ion binding ligand. For example, as shownin the Examples, the target substance may be the Ca+2 ion, and theblocking moiety may be a calcium binding ligand such as is known in theart (see Grynkiewicz et al., J. Biol. Chem. 260(6):3440-3450 (1985);Haugland, R. P., Molecular Probes Handbook of Fluorescent Probes andResearch Chemicals (1989-1991)). Other suitable target ions includeMn+2, Mg+2, Zn+2, Na+, and Cl-.

When Ca+2 is the target substance, preferred blocking moieties include,but are not limited to, the acetic acid groups ofbis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), ethyleneglycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA);ethylenediaminetetracetic acid (EDTA); and derivatives thereof, such asdisclosed in Tsien, Biochem. 19:2396-2404 (1980). Other known chelatorsof Ca+2 and other divalent ions, such as quin2(2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy]methyl-6-methoxy-8-[bis(carboxymethyl)amino]quinoline;fura-1, fura-2, fura-3, stil-1, stil-2 and indo-1 (see Grynkiewicz etal., supra).

As for the enzyme/substrate embodiments, the metabolite may beassociated with a particular disease or condition within an animal. Forexample, as outlined below, BAPTA-DOTA derivatives may be used todiagnose Alzeheimer's disease and other neurological disorders.

In a preferred embodiment, the blocking moiety is a ligand for acell-surface receptor or is a ligand which has affinity for aextracellular component. In this embodiment, as for the physiologicalagent embodiment, the ligand has sufficient affinity for the metal ionto prevent the rapid exchange of water molecules in the absence of thetarget substance. Alternatively, there may be R groups "locking" theligand into place, as described herein, resulting in either thecontribution of a coordination atom or that the ligand serves as acoordination site barrier. In this embodiment the ligand blocking moietyhas a higher affinity for the target substance than for the metal ion.Accordingly, in the presence of the target substance, the ligandblocking moiety will interact with the target substance, thus freeing upat least one coordination site in the metal ion complex and allowing therapid exchange of water and an increase in relaxivity. Additionally, inthis embodiment, this may result in the accumulation of the MRI agent atthe location of the target, for example at the cell surface. This may besimilar to the situation where the blocking moiety is an enzymeinhibitor, as well.

In a preferred embodiment, the blocking moiety is a photocleavablemoiety. That is, upon exposure to a certain wavelength of light, theblocking moiety is cleaved, allowing an increase in the exchange rate ofwater in at least one coordination site of the complex. This embodimenthas particular use in developmental biology fields (cell lineage,neuronal development, etc.), where the ability to follow the fates ofparticular cells is desirable. Suitable photocleavable moieties aresimilar to "caged" reagents which are cleaved upon exposure to light. Aparticularly preferred class of photocleavable moieties are theO-nitrobenzylic compounds, which can be synthetically incorporated intoa blocking moiety via an ether, thioether, ester (including phosphateesters), amine or similar linkage to a heteroatom (particularly oxygen,nitrogen or sulfur). Also of use are benzoin-based photocleavablemoieties. A wide variety of suitable photocleavable moieties is outlinedin the Molecular Probes Catalog, supra.

In a preferred embodiment, the compounds have a structure depicted belowin Structure 18, which depicts a nitrobenzyl photocleavable group,although as will be appreciated by those in the art, a wide variety ofother moieties may be used: ##STR7## Structure 18 depicts a DOTA-typechelator, although as will be appreciated by those in the art, otherchelators may be used as well. R₂₆ is a linker as defined below.Similarly, the X₂ group may be as defined above, although additionalstructures may be used, for example a coordination site barrier asoutlined herein. Similarly, there may be substitutent groups on thearomatic ring, as is known in the art.

The blocking moiety itself may block or occupy at least one coordinationsite of the metal ion. That is, one or more atoms of the blocking moiety(i.e. the enzyme substrate, ligand, moiety which interacts with aphysiological agent, photocleavable moiety, etc.) itself serves as acoordination atom, or otherwise blocks access to the metal ion by sterichinderance. For example, it appears that one or more of the atoms of thegalactose blocking moiety outlined in the Examples may be directcoordination atoms for the Gd(III) metal ion. Similarly, peptide basedblocking moieties for protease targets may contribute coordinationatoms.

In an alternative embodiment, the blocking moiety further comprises a"coordination site barrier" which is covalently tethered to the complexin such a manner as to allow disassociation upon interaction with atarget substance. For example, it may be tethered by one or more enzymesubstrate blocking moieties. In this embodiment, the coordination sitebarrier blocks or occupies at least one of the coordination sites of themetal ion in the absence of the target enzyme substance. Coordinationsite barriers are used when coordination atoms are not provided by thefunctional portion of the blocking moiety, i.e. the component of theblocking moiety which interacts with the target substance. The blockingmoiety or moieties such as an enzyme substrate serves as the tether,covalently linking the coordination site barrier to the metal ioncomplex. In the presence of the enzyme target, the enzyme cleaves one ormore of the enzyme substrates, either within the substrate or at thepoint of attachment to the metal ion complex, thus freeing thecoordination site barrier. The coordination site or sites are no longerblocked and the bulk water is free to rapidly exchange at thecoordination site of the metal ion, thus enhancing the image. As will beappreciated by those in the art, a similar result can be accomplishedwith other types of blocking moieties.

In one embodiment, the coordination site barrier is attached to themetal ion complex at one end, as is depicted in FIG. 1. When the enzymetarget cleaves the substrate blocking moiety, the coordination sitebarrier is released. In another embodiment, the coordination sitebarrier is attached to the metal ion complex with more than onesubstrate blocking moiety, as is depicted in FIG. 2 for two attachments.The enzyme target may cleave only one side, thus removing thecoordination site barrier and allowing the exchange of water at thecoordination site, but leaving the coordination site barrier attached tothe metal ion complex. Alternatively, the enzyme may cleave thecoordination site barrier completely from the metal ion complex.

In a preferred embodiment, the coordination site barrier occupies atleast one of the coordination sites of the metal ion. That is, thecoordination site barrier contains at least one atom which serves as atleast one coordination atom for the metal ion. In this embodiment, thecoordination site barrier may be a heteroalkyl group, such as an alkylamine group, as defined above, including alkyl pyridine, alkylpyrroline, alkyl pyrrolidine, and alkyl pyrole, or a carboxylic orcarbonyl group. The portion of the coordination site barrier which doesnot contribute the coordination atom may also be consider a linkergroup. Preferred coordination site barriers are depicted in FIG. 2.

In an alternative embodiment, the coordination site barrier does notdirectly occupy a coordination site, but instead blocks the sitesterically. In this embodiment, the coordination site barrier may be analkyl or substituted group, as defined above, or other groups such aspeptides, proteins, nucleic acids, etc.

In this embodiment, the coordination site barrier is preferrably linkedvia two enzyme substrates to opposite sides of the metal ion complex,effectively "stretching" the coordination site barrier over thecoordination site or sites of the metal ion complex, as is depicted inFIG. 2.

In some embodiments, the coordination site barrier may be "stretched"via an enzyme substrate on one side, covalently attached to the metalion complex, and a linker moeity, as defined below, on the other. In analternative embodiment, the coordination site barrier is linked via asingle enzyme substrate on one side; that is, the affinity of thecoordination site barrier for the metal ion is higher than that ofwater, and thus the blocking moiety, comprising the coordination sitebarrier and the enzyme substrate, will block or occupy the availablecoordination sites in the absence of the target enzyme.

In some embodiments, the metal ion complexes of the invention have asingle associated or bound blocking moiety. In such embodiments, thesingle blocking moiety impedes the exchange of water molecules in atleast one coordination site. Alternatively, as is outlined below, asingle blocking moiety may hinder the exchange of water molecules inmore than one coordination site, or coordination sites on differentchelators.

In alternative embodiments, two or more blocking moieties are associatedwith a single metal ion complex, to implede the exchange of water in atleast one or more coordination sites.

It should be appreciated that the blocking moieties of the presentinvention may further comprise a linker group as well as a functionalblocking moiety. That is, blocking moieties may comprise functionalblocking moieties in combination with a linker group and/or acoordination site barrier.

Linker groups (sometimes depicted herein as R₂₆) will be used tooptimize the steric considerations of the metal ion complex. That is, inorder to optimize the interaction of the blocking moiety with the metalion, linkers may be introduced to allow the functional blocking moietyto block or occupy the coordination site. In general, the linker groupis chosen to allow a degree of structural flexibility. For example, whena blocking moiety interacts with a physiological agent which does notresult in the blocking moiety being cleaved from the complex, the linkermust allow some movement of the blocking moiety away from the complex,such that the exchange of water at at least one coordination site isincreased.

Generally, suitable linker groups include, but are not limited to, alkyland aryl groups, including substituted alkyl and aryl groups andheteroalkyl (particularly oxo groups) and heteroaryl groups, includingalkyl amine groups, as defined above. Preferred linker groups includep-aminobenzyl, substituted p-aminobenzyl, diphenyl and substituteddiphenyl, alkyl furan such as benzylfuran, carboxy, and straight chainalkyl groups of 1 to 10 carbons in length. Particularly preferredlinkers include p-aminobenzyl, methyl, ethyl, propyl, butyl, pentyl,hexyl, acetic acid, propionic acid, aminobutyl, p-alkyl phenols,4-alkylimidazole. The selection of the linker group is generally doneusing well known molecular modeling techniques, to optimize theobstruction of the coordination site or sites of the metal ion. Inaddition, as outlined in the Examples, the length of this linker may bevery important in order to achieve optimal results. As shown in FIG. 11,the length of the linker, i.e the spacer between the chelator and thecoordination atom(s) of the blocking moiety, contributes to the stericconformation and association of the coordination atoms with the metalion, thus allowing excellent blocking of the metal ion by the blockingmoiety.

The blocking moiety is attached to the metal ion complex in a variety ofways. In a preferred embodiment, as noted above, the blocking moiety isattached to the metal ion complex via a linker group. Alternatively, theblocking moiety is attached directly to the metal ion complex; forexample, as outlined below, the blocking moiety may be a substituentgroup on the chelator.

In a preferred embodiment at least one of the R groups attached to the"arms" of the chelator, for example R₉, R₁₀, R₁₁ or R₁₂ of the DOTAstructures, or R₁₃, R₁₄, R₁₇, R₂₀ or R₂₁ of the DTPA structures,comprises an alkyl (including substituted and heteroalkyl groups), oraryl (including substituted and heteroaryl groups), i.e. is a groupsterically bulkier than hydrogen. This is particular useful to drive theequilibrium towards "locking" the coordination atom of the arm intoplace to prevent water exchange, as is known for standard MRI contrastagents. Preferred groups include the C1 through C6 alkyl groups withmethyl being particularly preferred.

This is particularly preferred when the blocking moiety is attached viaone of the "arms", for example when a blocking moiety is at position X₁to X₄ (Structure 6), position S, T, U or V (Structure 8) or position H,I, J or K of Structure 16.

However the inclusion of too many groups may drive the equilibrium inthe other direction effectively locking the coordination atom out ofposition, as is shown in Example 3. Therefore in a preferred embodimentonly 1 or 2 of these positions is a non-hydrogen group, unless othermethods are used to drive the equilibrium towards binding.

The blocking moieties are chosen and designed using a variety ofparameters. In the embodiment which uses a coordination site barrier,i.e. when the functional group of the blocking moiety does not provide acoordination atom, and the coordination site barrier is fastened orsecured on two sides, the affinity of the coordination site barrier ofthe blocking moiety for the metal ion complex need not be great, sinceit is tethered in place. That is, in this embodiment, the complex is"off" in the absence of the target substance. However, in the embodimentwhere the blocking moiety is linked to the complex in such a manner asto allow some rotation or flexibility of the blocking moiety, forexample, it is linked on one side only, such as the galactose embodimentof the examples, the blocking moiety should be designed such that itoccupies the coordination site a majority of the time. Thus, forexample, the galactose-DOTA structure of Example 1 gives roughly a 20%increase in the signal in the presence of galactosidase, thus indicatingthat the galactose blocking moiety is in equilibrium between blocking oroccupying the coordination site and rotating free in solution. However,as described herein and shown in Example 3, these agents may be "locked"off using R groups on the carboxylic acid "arms" of a chelator, toreduce the rotational freedom of the group and thus effectively drivethe equilibrium to the "off" position, and thus result in a largerpercentage increase in the signal in the presence of the target.

When the blocking moiety is not covalently tethered on two sides, as isdepicted in FIG. 1, it should be understood that blocking moieties andcoordination site barriers are chosen to maximize three basicinteractions that allow the blocking moiety to be sufficientlyassociated with the complex to hinder the rapid exchange of water in atleast one coordination site of the complex. First, there may beelectrostatic interactions between the blocking moiety and the metalion, to allow the blocking moiety to associate with the complex.Secondly, there may be Van der Waals and dipole-dipole interactions.Thirdly, there may be ligand interactions, that is, one or morefunctionalities of the blocking moiety may serve as coordination atomsfor the metal. In addition, linker groups may be chosen to force orfavor certain conformations, to drive the equilibrium towards anassociated blocking moiety. Similarly, removing degrees of fredom in themolecule may force a particular conformation to prevail. Thus, forexample, the addition of alkyl groups, and particularly methyl groups,at positions equivalent to the R₉ to R₁₂ positions of Structure 7 whenthe blocking moiety is attached at W, X, Y or Z, can lead the blockingmoiety to favor the blocking position. Similar restrictions can be madein the other embodiments, as will be appreciated by those in the art.

Furthermore, effective "tethering" of the blocking moiety down over themetal ion may also be done by engineering in other non-covalentinteractions that will serve to increase the affinity of the blockingmoiety to the chelator complex, as is depicted below.

Potential blocking moieties may be easily tested to see if they arefunctional; that is, if they sufficiently occupy or block theappropriate coordination site or sites of the complex to prevent rapidexchange of water. Thus, for example, complexes are made with potentialblocking moieties and then compared with the chelator without theblocking moiety in imaging experiments. Once it is shown that theblocking moiety is a sufficient "blocker", the target substance is addedand the experiments repeated, to show that interaction with the targetsubstance increases the exchange of water and thus enhances the image.

Thus, as outlined above, the metal ion complexes of the presentinvention comprise a paramagnetic metal ion bound to a chelator and atleast one blocking moiety. In a preferrred embodiment, the metal ioncomplexes have the formula shown in Structure 6: ##STR8## In Structure6, M is a paramagnetic metal ion selected from the group consisting ofGd(III), Fe(III), Mn(II), Yt(III), and Dy(III). A, B, C and D are eacheither single or double bonds. The R₁ through R₁₂ groups are alkyl oraryl groups, as defined above, including substituted alkyl and arylgroups, phosphorus groups, or a blocking moiety, as described above. X₁through X₄ are --OH, --COO--, --(CH2)_(n) OH (with --CH₂ OH beingpreferred), --(CH2)_(n) COO-- (with CH₂ COO-- being preferred) or ablocking moiety. n is from 1 to 10, with from 1 to 5 being preferred. Atleast one of R₁ to R₁₂ and X₁ to X₄ is a blocking moiety.

Structure 6 includes Structures 7 and 8, shown below: ##STR9## In thisembodiment, W, X, Y and Z are as defined above for X, and at least oneof the R₁ to R₁₂ groups is a blocking moiety.

As applied to DOTA, the four nitrogens of the DOTA ring, and the W, X, Yand Z groups provide 8 of the coordination atoms for the paramagneticmetal ion. The ninth coordination atom is provided by a blocking moietywhich is substituted at one of the R₁ to R₁₂ positions. In a preferredembodiment, the other R groups are either hydrogen or methyl; in aparticularly preferred embodiment the chelator is Gd-MCTA, which has asingle methyl group on the DOTA ring (see Meyer et al., Invest. Radiol.25:S53 (1990)).

In an alternative embodiment, the metal ion complexes have the formuladepicted in Structure 8: ##STR10## In this embodiment, S, T, U, and Vare --OH, --COO--, --(CH2)_(n) OH (with --CH₂ OH being preferred),--(CH2)_(n) COO-- (with CH₂ COO-- being preferred) or a blocking moiety.In this embodiment, the four nitrogens of the DOTA ring, and three ofthe S, T, U or V groups provide 7 of the coordination atoms for theparamagnetic metal ion. The remaining coordination atoms are provided bya blocking moiety which is substituted at one of the S, T, U or Vpositions. Alternatively, the coordination sites are either filled bycoordination atoms provided by the S, T, U or V groups, or blocked bythe S, T, U or V structure, or both. In addition, Structure 8 does notdepict the A, B, C and D bonds, but as for the other embodiments, thesebonds may be either single or double bonds.

As applied to DOTA, the four nitrogens of the DOTA ring, and the(generally) three S, T and U groups provide 7 of the coordination atomsfor the Gd(III) paramagnetic metal ion. The eigth and ninth coordinationatoms are provided by a blocking moiety which is substituted at one ofthe S, T, U and V positions. As above, the other R groups are preferablyeither hydrogen or methyl, with Gd-MCTA being especially preferred.

In the Structures depicted herein, any or all of A, B, C or D may be asingle bond or a double bond. It is to be understood that when one ormore of these bonds are double bonds, there may be only a singlesubstitutent group attached to the carbons of the double bond. Forexample, when A is a double bond, there may be only a single R₁ and asingle R₂ group attached to the respective carbons; in a preferredembodiment, as described below, the R₁ and R₂ groups are hydrogen. In apreferred embodiment, A is a single bond, and it is possible to have twoR₁ groups and two R₂ groups on the respective carbons. In a preferredembodiment, these groups are all hydrogen with the exception of a singleblocking moiety, but alternate embodiments utilize two R groups whichmay be the same or different. That is, there may be a hydrogen and ablocking group attached in the R₁ position, and two hydrogens, two alkylgroups, or a hydrogen and an alkyl group in the R₂ positions.

It is to be understood that the exact composition of the X₁ -X₄(Structure 6) S, T, U, V (Structure 8) or W, X, Y and Z (Structure 7)groups will depend on the presence of the metal ion. That is, in theabsence of the metal ion, the groups may be --OH, --COOH, --(CH₂)_(n)OH, or (CH₂)_(n) COOH; however, when the metal is present, the groupsmay be --OH, --COO--, --(CH₂)_(n) O--, or (CH₂)_(n) COO--.

In a preferred embodiment, the compositions have the formula shown inStructure 9: ##STR11## In this embodiment, there is a single blockingmoiety attached to the metal ion complex. That is, all but one of the Rgroups are hydrogen. It should be appreciated that the blocking moietymay be at any of the R positions.

In a preferred embodiment, the magnetic resonance imaging agents areused to detect Ca+2 ions, and have the structure depicted in Structure10: ##STR12## In this embodiment, the blocking moiety comprises a linkerand the BAPTA molecule, although any of the fura-type Ca⁺² ligands maybe substituted. Without being bound by theory, it appears that one ofthe carboxy groups of the BAPTA moiety serves to provide a coordinationatom in the absence of Ca+2. However, in the presence of Ca+2, thecarboxy group chelates Ca+2, and thus is unavailable as a coordinationgroup, thus allowing the rapid exchange of water. Preferably, the metalion is Gd(III), the R groups are all hydrogen, and the W, X, Y and Zgroups are carboxy.

In one embodiment the carboxylic acid groups of the BAPTA molecule maybe protected with acetate protecting groups, resulting a neutralmolecule that may then cross membranes. Once inside a cell,intracellular esterases can cleave off the acetate protecting groups,allowing the detection of Ca⁺². See Li et al., Tetrahedron53(35):12017-12040 (1997).

In a preferred embodiment, the compositions have the formula shown inStructure 11: ##STR13## In this embodiment, there is a single blockingmoiety attached to the metal ion complex. It should be appreciated thatthe blocking moiety may be at any of the S, T, U or V positions.Similarly, a single blocking moiety may be attached to DTPA.

In a preferred embodiment, the magnetic resonance imaging contrastagents have the structure shown in Structure 12: ##STR14## In thisembodiment, the blocking moiety comprises a linker and a carbohydrate,attached to the complex via a β(1, 4) linkage such as is recognized bylactose or β-galactosidase. Without being bound by theory, it isapparent that the galactose moiety provides a coordination atom, suchthat in the absence of β-galactosidase there is reduced exchange ofwater in the complex. Upon exposure to β-galactosidase, the carbohydrateblocking moiety is cleaved off, removing the coordination atom andallowing the rapid exchange of water. Preferably, the R groups arehydrogen, and the W, X, Y and Z groups are carboxy.

In another embodiment, the metal ion complexes have the formula depictedin Structure 13: ##STR15## In this embodiment, R₂₂, R₂₃ and R₂₄ comprisea blocking moiety, with R₂₃ being a coordination site barrier which alsoserves to contribute a coordination atom. It is to be understood thatthe R₂₂ and R₂₄ groups may be attached at any of the R₁ to R₁₂positions. Preferred R₂₃ groups include, but are not limited to,compounds listed above that provide a coordination atom, blockingmoieties, and those shown in FIG. 2. R₂₂ and R₂₄ may also comprise alinker, as defined above and as shown in Structure 14, below. PreferredR₂₂ and R₂₄ groups include enzyme substrates which are cleaved uponexposure to the enzyme, such as carbohydrates and peptides. Accordingly,when the target substance is a carbohydrase such as β-galactosidase, thecompositions have the formula shown in Structure 14: ##STR16## In thisembodiment, the blocking moiety comprises two linkers, twocarbohydrates, and a coordination site barrier. The carbohydrates areattached to the complex via a linkage which will be recognized by acarbohydrase such as a β(1, 4) linkage such as is recognized by lactoseor β-galactosidase. The R₂₂ group provides a coordination atom in theabsence of the carbohydrase such there is no rapid exchange of water inthe complex. Upon exposure to the carbohydrase, such as β-galactosidase,one or both of the carbohydrate blocking moieties are cleaved off,removing the coordination atom and allowing the rapid exchange of water.Preferably, the R groups are hydrogen, and the W, X, Y and Z groups arecarboxy. Alternatively, the blocking moiety could comprise peptides fora protease target substance.

In place of the carbohydrates in Structure 14, an alternative embodimentutilizes peptides. That is, a peptide comprising 2 to 5 amino acids oranalogs may be "stretched" from one side of the complex to the other,and linker groups may or may not be used. Similarly, nucleic acids maybe used.

Alternatively, there may not be covalent attachment at both ends. Asdiscussed above, effective "tethering" of the blocking moiety down overthe metal ion may also be done by engineering in other non-covalentinteractions that will serve to increase the affinity of the blockingmoiety to the chelator complex. Thus, for example, electrostaticinteractions may be used, as is generally depicted below for a DOTAderivative in Structure 15: ##STR17## In Structure 15, the blockingmoeity/coordination site barrier occupies the X₃ position, although anyposition may be utilized. E₁ and E₂ and electrostatic moieties bearingopposite charges. In Structure 15, the E₂ group is shown a position R₈,although any position may be used.

A further embodiment utilizes metal ion complexes having the formulashown in Structure 16: ##STR18## It is to be understood that, as above,the exact composition of the H, I, J, K and L groups will depend on thepresence of the metal ion. That is, in the absence of the metal ion, H,I, J, K and L are --OH, --COOH, --(CH₂)_(n) OH, or (CH₂)_(n) COOH;however, when the metal is present, the groups are --OH, --COO--,--(CH₂)_(n) OH, or (CH₂)_(n) COO--.

In this embodiment, R₁₃ through R₂₁ are alkyl or aryl, includingsubstituted and hetero derivatives, a phosphorus moiety or a blockingmoiety, all as defined above. In a preferred embodiment, R₁₂ to R₂₁ arehydrogen. At least one of R₁₃ -R₂₁, H, I, J, K or L is a blockingmoiety, as defined above.

In a preferred embodiment, the MRI contrast agents of the inventioncomprise more than one metal ion, such that the signal is increased. Asis outlined below, this may be done in a number of ways, some of whichare shown in FIG. 10.

In a preferred embodiment, the MRI agents of the invention comprise atleast two paramagnetic metal ions, each with a chelator and blockingmoiety; that is, multimeric MRI agents are made. In a preferredembodiment, the chelators are linked together, either directly orthrough the use of a linker such as a coupling moiety or polymer. Forexample, using substitution groups that serve as functional groups forchemical attachment on the chelator, attachment to other chelators maybe accomplished. As will be appreciated by those in the art, attachmentof more than one MRI agent may also be done via the blocking moieties(or coordination site barriers, etc.), although these are generally notpreferred.

In a preferred embodiment, the chelators of the invention include one ormore substitution groups that serve as functional groups for chemicalattachment. Suitable functional groups include, but are not limited to,amines (preferably primary amines), carboxy groups, and thiols(including SPDP, alkyl and aryl halides, maleimides, α-haloacetyls, andpyridyl disulfides) are useful as functional groups that can allowattachment.

In one embodiment, the chelators are linked together directly, using atleast one functional group on each chelator. This may be accomplishedusing any number of stable bifunctional groups well known in the art,including homobifunctional and heterobifunctional linkers (see PierceCatalog and Handbook, 1994, pages T155-T200, hereby expresslyincorporated by reference). This may result in direct linkage, forexample when one chelator comprises a primary amine as a functionalgroup and the second comprises a carboxy group as the functional group,and carbodiimide is used as an agent to activate the carboxy for attachby the nucleophilic amine (see Torchilin et al., Critical Rev.Therapeutic Drug Carrier Systems, 7(4):275-308 (1991). Alternatively, aswill be appreciated by those in the art, the use of some bifunctionallinkers results in a short coupling moiety being present in thestructure. A "coupling moiety" is capable of covalently linking two ormore entities. In this embodiment, one end or part of the couplingmoiety is attached to the first MRI contrast agent, and the other isattached to the second MRI agent. The functional group(s) of thecoupling moiety are generally attached to additional atoms, such asalkyl or aryl groups (including hetero alkyl and aryl, and substitutedderivatives), to form the coupling moiety. Oxo linkers are alsopreferred. As will be appreciated by those in the art, a wide range ofcoupling moieties are possible, and are generally only limited by theability to synthesize the molecule and the reactivity of the functionalgroup. Generally, the coupling moiety comprises at least one carbonatom, due to synthetic requirements; however, in some embodiments, thecoupling moiety may comprise just the functional group.

In a preferred embodiment, the coupling moiety comprises additionalatoms as a spacer. As will be appreciated by those in the art, a widevariety of groups may be used. For example, a coupling moiety maycomprise an alkyl or aryl group substituted with one or more functionalgroups. Thus, in one embodiment, a coupling moiety containing amultiplicity of functional groups for attachment of multiple MRIcontrast agents may be used, similar to the polymer embodiment describedbelow. For example, branched alkyl groups containing multiple functionalgroups may be desirable in some embodiments.

In an additional embodiment, the linker is a polymer. In thisembodiment, a polymer comprising at least one MRI contrast agent of theinvention is used. As will be appreciated by those in the art, these MRIcontrast agents may be monomeric (i.e. one metal ion, one chelator, oneblocking moiety) or a duplex, as is generally described below (i.e. twometal ions, two chelators, one blocking moiety). Preferred embodimentsutilize a plurality of MRI agents per polymer. The number of MRI agentsper polymer will depend on the density of MRI agents per unit length andthe length of the polymer.

The character of the polymer will vary, but what is important is thatthe polymer either contain or can be modified to contain functionalgroups for the the attachment of the MRI contrast agents of theinvention. Suitable polymers include, but are not limited to,functionalized dextrans, styrene polymers, polyethylene and derivatives,polyanions including, but not limited to, polymers of heparin,polygalacturonic acid, mucin, nucleic acids and their analogs includingthose with modified ribose-phosphate backbones, the polypeptidespolyglutamate and polyaspartate, as well as carboxylic acid, phosphoricacid, and sulfonic acid derivatives of synthetic polymers; andpolycations, including but not limited to, synthetic polycations basedon acrylamide and 2-acrylamido-2-methylpropanetrimethylamine,poly(N-ethyl-4-vinylpyridine) or similar quarternized polypyridine,diethylaminoethyl polymers and dextran conjugates, polymyxin B sulfate,lipopolyamines, poly(allylamines) such as the strong polycationpoly(dimethyldiallylammonium chloride), polyethyleneimine, polybrene,spermine, spermidine and polypeptides such as protamine, the histonepolypeptides, polylysine, polyarginine and polyornithine; and mixturesand derivatives of these. Particularly preferred polycations arepolylysine and spermidine, with the former being especially preferred.Both optical isomers of polylysine can be used. The D isomer has theadvantage of having long-term resistance to cellular proteases. The Lisomer has the advantage of being more rapidly cleared from the subject.As will be appreciated by those in the art, linear and branched polymersmay be used.

A preferred polymer is polylysine, as the --NH₂ groups of the lysineside chains at high pH serve as strong nucleophiles for multipleattachment of activated chelating agents. At high pH the lysine monomersare coupled to the MRI agents under conditions that yield on average5-20% monomer substitution.

In some embodiments, particularly when charged polymers are used, theremay be a second polymer of opposite charge to the first that iselectrostatically associated with the first polymer, to reduce theoverall charge of polymer-MRI agent complex. This second polymer may ormay not contain MRI agents.

The size of the polymer may vary substantially. For example, it is knownthat some nucleic acid vectors can deliver genes up to 100 kilobases inlength, and artificial chromosomes (megabases) have been delivered toyeast. Therefore, there is no general size limit to the polymer.However, a preferred size for the polymer is from about 10 to about50,000 monomer units, with from about 2000 to about 5000 beingparticularly preferred, and from about 3 to about 25 being especiallypreferred.

It should be understood that the multimeric MRI agents of the inventionmay be made in a variety of ways, including those listed above. What isimportant is that manner of attachment does not significantly alter thefunctionality of the agents; that is, the agents must still be "off" inthe absence of the target substance and "on" in its presence.

In a preferred embodiment, the MRI contrast agents of the invention are"duplexes". In this embodiment, the MRI duplex comprises two chelators,each with a paramagnetic metal ion, and at least one blocking moietythat restricts the exchange of water in at least one coordination siteof each chelator. In this way, a sort of signal amplification occurs,with two metal ions increasing the signal with a single target molecule.While "duplex" implies two chelators, it is intended to refer tocomplexes comprising a single blocking moiety donating coordinationatoms to more than 1 metal ion/chelator complex. As will be appreciatedby those in the art, the MRI agents of this embodiment may have a numberof different conformations, as is generally shown in FIG. 10. As will beappreciated by those in the art, the R₂₆, R₂₇ and R₂₈ groups of thefigure can be attached to any of the positions described herein, to anyR groups or X₁ -X₄, S, T, U, V, W, X, Y, or Z groups.

As outlined above, the MRI duplex moieties may also be combined intohigher multimers, either by direct linkage or via attachment to apolymer.

In a preferred embodiment, the blocking moiety is BAPTA, as is generallydepicted below in Structure 17, with propyl linking groups between thechelators and the BAPTA derivative: ##STR19##

As will be appreciated by those in the art, the structure depicted inStructure 17 may be altered, for example, replacing the phenyl groups ofthe BAPTA derivative with cycloalkyl groups, or removing them entirely,as is generally depicted in Structure 19: ##STR20##

As noted above, the carboxylic acids of the BAPTA molecule may also beprotected using acetate protecting groups, to render a neutral moleculefor entry into cells, that then can be reactivated via cleavage byintracellular esterases.

In addition, although Structures 17 and 19 have ethylene groups betweenthe oxygens of the bridge of BAPTA, methylene and propylene may also beused, as well as substituted derivatives of these.

In a preferred embodiment, A, B, C and D are single bonds, R₁ -R₁₂ arehydrogen, and each R₂₆ is --CH₂ O--, with the CH₂ group being attachedto the macrocycle.

In addition, the complexes and metal ion complexes of the invention mayfurther comprise one or more targeting moieties. That is, a targetingmoiety may be attached at any of the R positions (or to a linker,including a polymer, or to a blocking moiety, etc.), although in apreferred embodiment the targeting moiety does not replace acoordination atom. By "targeting moiety" herein is meant a functionalgroup which serves to target or direct the complex to a particularlocation or association. Thus, for example, antibodies, cell surfacereceptor ligands and hormones, lipids, sugars and dextrans, alcohols,bile acids, fatty acids, amino acids, and peptides may all be attachedto localize or target the contrast agent to a particular site.

In a preferred embodiment, the metal ion complexes of the presentinvention are water soluble or soluble in aqueous solution. By "solublein aqueous solution" herein is meant that the MRI agent has appreciablesolubility in aqueous solution and other physiological buffers andsolutions. Solubility may be measured in a variety of ways. In oneembodiment, solubility is measured using the United States Pharmacopeiasolubility classifications, with the metal ion complex being either verysoluble (requiring less than one part of solvent for 1 part of solute),freely soluble (requiring one to ten parts solvent per 1 part solute),soluble (requiring ten to thirty parts solvent per 1 part solute),sparingly soluble (requiring 30 to 100 parts solvent per 1 part solute),or slightly soluble (requiring 100-1000 parts solvent per 1 partsolute).

Testing whether a particular metal ion complex is soluble in aqueoussolution is routine, as will be appreciated by those in the art. Forexample, the parts of solvent required to solubilize a single part ofMRI agent may be measured, or solubility in gm/ml may be determined.

The complexes of the invention are generally synthesized using wellknown techniques. See, for example, Moi et al., supra; Tsien et al.,supra; Borch et al., J. Am. Chem. Soc., p2987 (1971); Alexander, (1995),supra; Jackels (1990), supra, U.S. Pat. Nos. 5,155,215, 5,087,440,5,219,553, 5,188,816, 4,885,363, 5,358,704, 5,262,532; Meyer et al.,(1990), supra, Moi et al., (1988), and McMurray et al., BioconjugateChem. 3(2):108-117 (1992)).

For DOTA derivatives, the synthesis depends on whether nitrogensubstitution or carbon substitution of the cyclen ring backbone isdesired. For nitrogen substitution, such as is exemplified by thegalactose-DOTA structures of the examples, the synthesis begins withcyclen or cyclen derivatives, as is well known in the art; see forexample U.S. Pat. Nos. 4,885,363 and 5,358,704. FIGS. 3 and 4 depict thenitrogen substitution as exemplified by galactose-DOTA derivatives.

For carbon substitution, such as is exemplified by the BAPTA-DOTAstructures of the examples, well known techniques are used. See forexample Moi et al., supra, and Gansow, supra. FIGS. 5 and 6 depict thecarbon substitution as exemplified by the BAPTA-DOTA type derivatives.

The contrast agents of the invention are complexed with the appropriatemetal ion as is known in the art. While the structures depicted hereinall comprise a metal ion, it is to be understood that the contrastagents of the invention need not have a metal ion present initially.Metal ions can be added to water in the form of an oxide or in the formof a halide and treated with an equimolar amount of a contrast agentcomposition. The contrast agent may be added as an aqueous solution orsuspension. Dilute acid or base can be added if need to maintain aneutral pH. Heating at temperatures as high as 100° C. may be required.

The complexes of the invention can be isolated and purified, for exampleusing HPLC systems.

Pharmaceutical compositions comprising pharmaceutically acceptable saltsof the contrast agents can also be prepared by using a base toneutralize the complexes while they are still in solution. Some of thecomplexes are formally uncharged and do not need counterions.

Once synthesized, the metal ion complexes of the invention have use asmagnetic resonance imaging contrast or enhancement agents. Specifically,the functional MRI agents of the invention have several important uses.First, they may be used to diagnose disease states of the brain, as isoutlined below. Second, they may be used in real-time detection anddifferentiation of myocardial infraction versus ischemia. Third, theymay be used in in vivo, i.e. whole organism, investigation of antigensand immunocytochemistry for the location of tumors. Fourth, they may beused in the identification and localization of toxin and drug bindingsites. In addition, they may be used to perform rapid screens of thephysiological response to drug therapy.

The metal ion complexes of the invention may be used in a similar mannerto the known gadolinium MRI agents. See for example, Meyer et al.,supra; U.S. Pat. No. 5,155,215; U.S. Pat. No. 5,087,440; Margerstadt etal., Magn. Reson. Med. 3:808 (1986); Runge et al., Radiology 166:835(1988); and Bousquet et al., Radiology 166:693 (1988). The metal ioncomplexes are administered to a cell, tissue or patient as is known inthe art. A "patient" for the purposes of the present invention includesboth humans and other animals and organisms, such as experimentalanimals. Thus the methods are applicable to both human therapy andveterinary applications. In addition, the metal ion complexes of theinvention may be used to image tissues or cells; for example, see Aguayoet al., Nature 322:190 (1986).

Generally, sterile aqueous solutions of the contrast agent complexes ofthe invention are administered to a patient in a variety of ways,including orally, intrathecally and especially intraveneously inconcentrations of 0.003 to 1.0 molar, with dosages from 0.03, 0.05, 0.1,0.2, and 0.3 millimoles per kilogram of body weight being preferred.Dosages may depend on the structures to be imaged. Suitable dosagelevels for similar complexes are outlined in U.S. Pat. Nos. 4,885,363and 5,358,704.

In addition, the contrast agents of the invention may be delivered viaspecialized delivery systems, for example, within liposomes (see Navon,Magn. Reson. Med. 3:876-880 (1986)) or microspheres, which may beselectively taken up by different organs (see U.S. Pat. No. 5,155,215).

In some embodiments, it may be desirable to increase the blood clearancetimes (or half-life) of the MRI agents of the invention. This has beendone, for example, by adding carbohydrate polymers to the chelator (seeU.S. Pat. No. 5,155,215). Thus, one embodiment utilizes polysaccharidesas substitution R groups on the compositions of the invention.

A preferred embodiment utilizes complexes which cross the blood-brainbarrier. Thus, as is known in the art, a DOTA derivative which has oneof the carboxylic acids replaced by an alcohol to form a neutral DOTAderivative has been shown to cross the blood-brain barrier. Thus, forexample, neutral complexes are designed that cross the blood-brainbarrier with blocking moieties which detect Ca+2 ions. These compoundsare used in MRI of a variety of neurological disorders, includingAlzeheimer's disease. Currently it is difficult to correctly diagnosisAlzeheimer's disease, and it would be useful to be able to have aphysiological basis to distinguish Alzeheimer's disease from depression,or other treatable clinical symptoms for example.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.The references cited herein are expressly incorporated by reference.

EXAMPLES Example 1 Synthesis and Characterization of Galactose-DOTADerivative

Synthesis of Do3a-hydroxyethyl-beta-galactose Gadolinium complex (FIG.4). Acetyl protected bromo-galactose (Aldrich) was reacted withbromoethanol. Difference ratios of the alpha- and beta-bromoethyl etherof the acetylgalactose were obtained in good yield. The isomers wereseparated using silica gel chromatography and their assigments were madeby hydrolyzing the acetyl protecting groups and comparing the proton NMRcoupling constants to known compounds. Recently an x-ray structure wasdone confirming these assignments (data not shown).

The beta-isomer was reacted with cyclen at reflux in chloroform withmonitoring of the reaction by TLC. Hydrolysis of the acetates wasacheived with TEA/MCOH/H₂ O overnight, and the solvent was removed underlow vacuum. The resulting product was reacted directly with bromoaceticacid and then maintained at pH 10-10.5 until the pH remained constant.The possible products all would have different charges in ammoniaacetate buffer and thus were separated by anion exchange chromatography.An ammonium acetate buffer gradient was used during FPLC anion exchangeto elute the desired compound, with detection at 218 nm. Gadoliniumoxide in water at 80° C. was used to insert the metal into the complex.The reaction was followed using fluorescence spectroscopy. The productwas purified by HPLC reverse phase chromatography using fluorescencespectroscopy for detection and the structure was confirmed using highresolution mass spectrometry. The overall yield for this essentially onepot synthesis was greater than 25%.

Synthesis of aceto-1-ethylbromo-β-galactose (FIG. 3): 1-Bromoethane-2-olwas reacted with 2,3,4,6-aceto-1 -a-bromo-galactose to produce a mixtureof α and β anomers (10/90) of aceto-1-ethylbromo-β-galactose in 68%yield (8.3g). The purified β anomer could be obtained using flashchromatography. Stereochemical assignments were made via a X-ray crystalstructure of the β anomer.

Aceto-1-ethylbromo-β-galactose was reacted with cyclen (Aldrich ChemicalCo.) to produce the monosubsituted product. The acetate protectinggroups were cleaved and the 3 carboxylic acid substituents were addedusing bromoacetic acid at pH 10.5. The product was isolated by anionexchange fast performance liquid chromatography (FPLC) observed byfluorescence spectrascopy in 37% yield. Gd³⁺ or Tb³⁺ was inserted intothe complexes and were purified using repeated collections on a reversephase HPLC analytic C₁₈ column with a water/acetonitrile gradient(0-10%) as the elute and fluorescence for detection (274 nm-ex and 315nm-em) in 70% yield. High resolution mass spectrum analysis of the solidprovided a parent molecular ion for the (M+Na)⁺ which exhibited thecorrect exact mass and the predicted isotope ratios.

Alternate synthetic route: Do3a methyl ester was synthesized byliterature methods. Do3a methyl ester was reacted with beta-bromoethylether of the acetylgalactose obtained as described in D₂ O/d4 methanolwhile maintaining the reaction at basic pH. The reaction was followed byNMR. First the acetate methyl ester cleaved and the sugar became watersoluble as judged by allowing the methanol to evaporate. Next the methylester was absorbed to cleave and finally at around pH 10 a shiftconsistant with the formation of the sugar Do3a was observed.

Summary of the synthesis of Do2a-hydroxyethyl-di-beta-galactose: Thereaction of cyclen with beta-bromoethyl ether of the acetylgalactose inchloroform was done. The reaction mixture was purified using silica gelchromatography. While the alpha isomer gave monosubstitution onlydi-substituted products were obtained for the beta isomer as shown inFIG. 5. The acetic acid derived arm was added as described for themonosubstituted compound above and purified by FPLC cation exchangeusing an acid water gradient. Individual fractions were detected by TLCspotting.

Characterization: The ability of β-galactosidase to remove thegalactopyranosal blocking group from GadGal was examined by HPLC. Thecleavage reaction was monitored using the distinct retention times ofthe complex and the complex without the galactopyranose residue. Uponincubation with native β-galactosidase, a peak with an elution time of15 min appeared that corresponds to the complex without galactopyranose.In a control experiment, using heat-inactivated β-galactosidase, theretention time of the peak remained constant. Thus, the HPLC experimentsconfirm the enzymatic processing of the complex by native but notheat-treated enzyme.

The effect the presence of the galactopyranosal residue on the waterexchange rate of the complex was tested by measuring the fluorescencespectra of the terbium (Tb³⁺) derivative (545 nm) in water/deuteriumoxide mixtures. Terbium was substituted for Gd because of the moreintense fluorescence signal and long lifetime when chelated. Thefluorescence of the terbium complex is quenched by H₂ O but not by D₂ O.This effect occurs because the excited state of the terbium is coupledto the OH oscillator but not the OD oscillator. Therefore, the lifetimeof the fluorescence signal is longer in D₂ O than in H₂ O. A plot of1/lifetime versus the percentage of H₂ O allows the calculation of thenumber of water molecules, q, that are fast exchange with the complex(Kumar et al., Pure and Appl. Chem. 65:515-520 (1993); Lie et al., J.Am. Chem. Soc. 117:8123-8138 (1995); Zhang et al., Inorg. Chem.31:5597-5600 (1992)). The q values for the terbium complexes in thepresence and absence of the galactopyranose were 0.7 and 1.2,respectively. Therefore, spectrofluorimetry confirms that thegalactopyranose blocking group hinders the fast exchange of water.

The effect of the enzymatic cleavage of the galactopyranose on the T₁ ofthe complex was assessed using NMR spectroscopy. The molar quantityrelated to these T₁ values is the relaxivity, R. R values at 500 MHzwere determined for the complex plus galactose (1800 mM s⁻¹) and minusgalactose (2400 mM s⁻¹) and compared to that of the related speciesProhance (2700 mM s⁻¹). The difference in observed relaxivity parallelsthe results obtained from the T₁ measurements for complexes. Theincrease in water exchange, demonstrated in the spectrofluorimetryexperiments, suggested that the T₁ of a solution of the agent shoulddecrease upon enzymatic processing. A 20% difference between themeasured T₁ values in the presence and absence of β-galactosidaseconfirmed this prediction. The complex exposed to β-galactosidase at twodifferent concentrations showed identical and significant decreases inthe solution T₁ A 20% change in observed T₁ accompanies cleavage of thegalactopyranose from the complex, consistent with the change in measuredhydration number, q, obtained from fluorescence measurements. Controlsolutions of the complex together with heat inactivated enzyme show nodecrease; in fact, the T₁ appeared to increase slightly. MRI microscopywas used to examine if the observed difference in T₁ between the complexin the presence and absence of the galactose would be sufficient toserve as a MRI contrast agent. Images obtained using a standardinversion recovery sequence revealed that the T₁ change generated byenzymatic processing could be visualized in a MR image (FIG. 9). Thecomplex was placed in 1.5-1.8 mm capillary tubes, either with or withoutβ-galactosidase. The images displayed in FIG. 9 show that the T₁mediated contrast was altered by the action of β-galactosidase, yieldingthe expected increase in the image contrast.

Example 2 Synthesis of BAPTA-DTPA and BAPTA-DOTA Derivatives

Two representative synthetic schemes are shown for the synthesis of aBAPTA-DTPA derivative in FIGS. 7 and 8. In FIG. 7 (the preferredmethod), structure I was prepared by modification of publishedprocedures (Tsien et al., supra) and coupled to hexamehtylenediaamineusing NaCNBrH₃ in dry methanol. The ratio of reactants used was 6:1:0.6(diamine:BAPTA aldehyde:NaCNBrH₃). The reaction was quenched with theaddition of concentrated HCl and the product purified by HPLC (II). Thismaterial was reacted with the mono (or bis) anhydride of DTPA with theprotecting groups left on the BAPTA until after the Gd(III)Cl₃ or Gd₂ O₃was added (elevated pH, heat). The final product was purified byion-exchange HPLC.

In FIG. 8, the monoanhydride of DTPA was prepared and reacted with abisalkylamine (e.g. NH₂ (CH₂)₆ (NH₂). This material was purified byion-exchange HPLC and placed in a round bottom flask equipped with argoninlet and pressure equalizing funnel. The BAPTA aldehyde in dry methanolwas added dropwise to a solution of alkylamine-DTPA in dry methanol and6 equivalents of HCl:MeOH was added. The reaction mixture was purifiedby HPLC, Gd(III) inserted as above, and the protecting groups removed byliterature procedures.

Example 3 The Use of R Groups to Increase Signal

The Example 1 compound exhibits an enhancement of roughly 20% uponexposure to the target analyte, in this case β-galactosidase. In orderto increase the MR contrast enhancement, our intention was to furtherdecrease the access of bulk water to the Gd(III) site by stabilizing theposition of the galactopyranose unit on top of the macrocyclicframework. Several studies dealing with intramolecular dynamic processesin tetraazacarboxyclic macrocycles were recently reported (see Kang etal., Inorg. Chem. 36:2912 (1993); Aime et al., Inorg. Chem. 36:2095(1997); Pittet et al., J. Am. Chem. Soc. Dalton Trans. 1997, 895-900;Spirlet et al., J. Am. Chem. Soc. Dalton Trans. 1997, 497-500, all ofwhich are incorporated by reference. This work demonstrated thatintroducing α-methyl groups to the ethylenic groups of carboxyclic armsincreases the rigidity of the amino-carboxylate macrocyclic framework.We therefore added sterically bulky α-methyl groups to two distinctsites of the molecule to make two new compounds. The first, "EGADMe", isthe GADGAL of Example 1 with a single methyl group on the DOTA armcontaining the galactosyl blocking moiety. The second, "CarboxyMe", isthe GADGAL of Example 1 with three methyl groups on the other three DOTAarms, leaving the arm containing the galoctosyl blocking moiety alone.The final products EGadMe and CarboxyMe as well as the intermediateswere characterized by NMR- and mass spectrometry.

The successful and complete enzymatic cleavage of the galactopyranoseblocking group from EGadMe and CarboxyMe, respectively, was followed byTLC chromatography (C 18 reverse phase plates in 20 mM tris-acetate, 10mM EDTA buffer pH 7.0, 8% acetonitrile), to produce EGADMecl andCarboxyMecl. While 90% of the galactopyranose units were enzymaticallycleaved from EGadMe within 3 days in an aequous solution containing 0.5mM EgadMe and 5 μM β-galactosidase at 37° C., the same effect wasobserved for CarboxyMe within a period of 24 hrs under the sameconditions. This result implied that the galactopyranose unit ofCarboxyMe might be more exposed and accessible for the enzyme, thereforeleading to a higher cleavage rate.

The effect of the enzymatic cleavage of the galactopyranose unit fromEGadMe and CarboxyMe on relaxation time T1 was determined by NMRspectroscopy at 500 MHz and 24° C. Various aqueous solutions of 0.5 mMEGadMe and CarboxyMe, respectively, were prepared, containing either:(a) no enzyme; (b) heat inactivated 5 μM β-galactosidase that wastreated at 80° C. for 10 min; (c) 5 μM β-galactosidase where T1 wasmeasured immediately after mixing; or (d) 5 μM β-galactosidase that wasreacted with the complex for 3.5 days at 37° C. A remarkably differencebetween the T1 of solutions containing EGadMe and those containingEGadMecl is clearly obvious. In the presence of EGadMecl the T1 of waterprotons is enhanced by 55% with respect to solutions containing EGadMe.These results indicate that EGadMe is a highly effective, fuctional or"smart" MRI contrast agent. A large difference in T1 between uncleavedand cleaved states represents the crucial factor for successful in vivoapplications. Preliminary in vivo studies indicate that the compoundfulfils these high expectations.

Interestingly, for solutions (a)-(d) containing CarboxyMe no significantvariations in TI were detected. However, for all CarboxyMe solutions thedetermined T1 values compare well to those obtained for solutionscontaining EGadMecl. Since the relaxation time of water protons is inthe same order of magnitude for CarboxyMe and CarboxyMecl it must beassumed that the galactopyranose unit does not block the Gd(III) site.It is therefore not effective in limiting the access of bulk water tothe metal site.

Molecular modeling studies support this hypothesis. The calculatedconfigurations of EGadMe and CarboxyMe were evaluated. In EGadMe thegalactopyranose unit is placed on top of the macrocyclic framework,thereby shielding the metal center. When the galactopyranose unit iscleaved off, the metal site becomes readily exposed and accessible forbulk water molecules to complete the Gd(III) coordination sphere.However, with CarboxyMe the galactopyranose unit is facing away from themacrocyclic unit instead of being located on top of it. The stericinfluence of the a-methyl groups on the carboxyclic arms seems toprevent the galactopyranose unit from taking a position on top of theGd(III) site. Therefore the metal site is easily accessible for bulkwater molecules in the uncleaved state as much as in the cleaved state,leading to comparable T1 data for both structures. Furthermore, a massspectrum obtained for CarboxyMe reveals that two chloride anions arecoordinated to the molecule that complete the two vacant Gd(III)coordination sites. In EGadMe these coordination sites are filled by thegalactopyranose unit.

To determine the efficiency of the galactopyranose unit in blocking theaccess of water molecules to Gd(III), thereby monitoring the waterexchange rate of EGadMe, the lifetime of the fluorescence signal of thecorresponding terbium derivative (ETbMe) was investigated. Thefluorescence of the terbium complex is quenched by H₂ O, since terbiumis strongly coupled to the OH oscillator. This effect is not observedfor the OD-oscillator. As a consequence, the lifetime of thefluorescence signal is longer in D₂ O than in H₂ O. Measuring ETbMe(lex=460 nm, lem=545 nm) in various H₂ O/D₂ O mixtures and plotting theresulting lifetimes vs. the D₂ O concentration leads to the number ofwater molecules q, that are in fast exchange with the complex (see Kumaret al., Pure Appl. Chem 65:515-520 (1993); Li et al, J. Am. Chem. Soc.117:8132 (1995); Horrocks et al., J. Am. Chem. Soc. 101:334 (1979);Zhang et al., Iorg. Chem. 31:5597 (1992), all of which are incorporatedby reference. For ETbMe a value of q=0.6 was determined, which wascompared to a value of q=1.2 observed for the tetraaceticacid macrocycleGd-DOTA (see Lauffer, Chem. Rev. 901-927 (1997). The change in thenumber of coordinating water molecules is clearly obvious. For thecorresponding cleaved complex, ETbMecl, the exponential decay of thefluorescence signal was much faster, indicating a number of q<1.However, attempts to detemine q for ETbMecl correctly were limited bythe time-resolution of the fluorimeter.

Assuming a number of 1<q<2 for EGadMecl is in total agreement with theT1 data observed for EGadMe and EGadMecl and structural arrangements,where the hydroxy group in EGadMecl is not tightly coordinating themetal site. Theory predicts that an increase in q is related to anincrease in T1. It can therefore be assumed that the large difference inT1 of 55% is a synergetic effect, i.e. effective blocking the access ofbulk water to the metal site by the galactopyranose unit in EGadMe andassuming a number of water molecules that are in fast exchange with thecomplex 1<q<2 in EGadMecl.

Example 4 Synthesis of Gd³⁺ -BAPTA-DO3A₂ ("CalGad")

The synthetic scheme for Calgad is shown in FIG. 14.

Compound 1: 2-Nitroresorcinol (2 g, 12.9 mmol) was dissolved in 95%ethanol (15 mL), 1 equivalent of NaOH was added slowly. After theaddition the solvent was removed under vacuum arid the resulting solidwas redissolved in 4 mL DMF with 1 equivalent of 3-bromopropanol. Afterheating the solution at 100 ° C. for 7 hours, the reaction was quenchedwith a few drops of acetic acid. After removing the solvent undervacuum, the residue was suspended in methylene chloride and filtered.Flash chromotography (CH₂ Cl₂ /MeOH, 20:1) afforded 1.08g (42%) of 1. ¹H-NMR (300 MHz, CDCl₃): 2.14(m, 2H, CH₂ CH₂ CH₂), 3.94(t, 2H, CH₂ OH),4.26(t, OCH₂), 6.6(d, 1H, aromatic H), 6.72(d, IH, aromatic H), 7.41(t,1H, aromatic H).

Compound 2: Compound 1 (0.8 g, 3.76 mmol) was dissolved in 8 mL DMF.1,2Dibromoethane (0.16 mL, 1.88 mmol) and K₂ CO3 (0.28 g) was then addedand the mixture was heated at 120° C. for 10 h. The reaction wasquenched with a few drops of acetic acid and the solvent was evaporatedunder vacuum. The residue was purified by flash chromatography (CH₂ Cl₂/MeOH, 20:1) and 0.46 g of product (55% ) was obtained. ¹ H-NMR (CDCl₃):2.0(m, 4H, CH₂ CH₂ CH₂), 3.85(m, 4H, CH₂ OH), 4.25(t, 4H, CH₂ O),4.46(s, 4H, OCH₂ CH₂ O), 6.66(m, 4H, aromatic H), 7.4(t, 2H, aromaticH).

Compound 3: Compound 2 (0.15 g) was suspended in a mixture of ethylacetate (10 mL) and 95% ethanol (10 mL). After adding Palladium catalyst(Pa/carbon, 10%, 50 mg), the solution was hydrogenated at 1 atmovernight. The catalyst was filtered off and the filtrate wasconcentrated under vacuum. The residue was used directly for the nextstep. Compound 4: The above residue was mixed with acetonitrile (2 mL),DIEA (0.25 mL, 1.37 mmol) and bromoethylacetate (0.15 mL, 1.37 mmol).The solution was refluxed under argon for 24 h and then cooled down toRT. Toluene (20 mL) was added to precipitate the DIEA salt. Afterfiltering off the precipitation, the filtrate was purified on flashchromotography (CH₂ Cl₂ /MeOH, 20:1) and 0.15 g of product (61% for 2steps) was obtained. MS (Electrospray) m/z (M+H)⁺, calcd 737 (C₃₆ H₅₃O₁₄ N₂), obsd 737.6, 759.4 (M+Na)⁺. ¹ H-NMR (CDCl₃): 1.25(t, 12H, CH₃),2.08(m, 4H, CH₂ CH₂ CH₂), 3.9(m, 4H, CH₂ OH), 4.05-4.4(m, 24H), 6.62(m,4H, aromatic H), 7.0(m, 2H, aromatic H).

Compound 5: Compound 4 (245 ma, 0.33 mmol), triphenylphosphine (262 ma,1 mmol) and carbon tetrabromide (332 ma, 1 mmol) were dissolved indiethyl ether (3 mL). After stirring at RT for 40 min, flashchromatography (CH₂ Cl₂ to CH₂ Cl₂ /MeOH, 20: 1) purification gave 0.19g of product (67%). ¹ H-NMR (CDCl₃): 1.21(t, 12H, CH₃), 2.34(m, 4H, CH₂CH₂ CH₂), 3.67(t, 4H, CH₂ Br), 4.05-4.34(m, 24H), 6.62(m,4H, aromaticH), 7.0(m, 2H, aromatic H).

Compound 6: Compound 5 (42 ma, 49 μmol) was reacted with cyclen (43 ma,0.25 mmol) in CHCl₃ (0.5 mL) for 30 hours. Flash chromatography (CHCl₃/MeOH/NH₃ H₂ O 12:4:1) afforded the product as a clear glass (41 ma,80%). MS (Electrospray) m/z (M+H)⁺, calcd 1046 (C₅₂ H₈₉ O,₂ N,o), obsd1046.0(M+H)⁺, 1067.8(M+Na)⁺, 1089.8(M+2Na-H)⁺, 523.4(M+2H)²⁺,534.4(M+H+Na)²⁺, ¹ H-NMR (CDCl₃): 1.2(t, 12H, CH₃), 2.0(br, 4H, CH₂ CH₂CH₂), 2.6-2.85(br. 36H), 4.0-4.4(br, 24H), 6.64(br, 4H. aromatic H),6.95(br, 2H, aromatic H).

Compound 7: Compound 6 (38 ma, 38 ,umol) was mixed with bromoacetic acid(37 ma, 266 μmol) in H₂ O (0.2 mL). Sodium hydroxide (SN) was slowlyadded to keep the pH of the solution above 10. When the pH of thesolution reached stable, the reaction was quenched with small amount ofacetic acid. The product was purified by reverse phase chromatography(LiChroprep RP-18, CH₃ CN/H₂ O, 5%-50%) and 38 mg (82%) of white powderwas obtained after lyophilization. MS (Electrospray) m/z (M+H)⁺, calcd1280 (C₅₆ H₈₃ N₁₀ O₂₄), obsd 1279.4(M-H)⁻, 639.3(M-2H)²⁻. ¹ H-NMR (D₂O): 2.32(br, 4H, CH₂ CH₂ CH₂), 3.05-3.83(br, 48H), 4.05(s, 8H), 4.27(br,4H), 4.7(s, 4H), 6.8(d, 2H, aromatic H), 6.95(d, 2H, aromatic H), 7.4(t,2H, aromatic H)

Gd3⁺ -complex of compound 7: The above ligand (compound 6, 16.5 ma, 12.9μmol) was dissolved in H₂ O (0.5 mL) containing GdCl3 (10.6 ma, 28.4μmol). NaOH (IN) was slowly added to keep the pH around 5˜6. The pH ofthe solution reached stable within 2 h indicating the completion of thereaction. The mixture was passed through a column packed with the Chelexresin (Biorad, Chelex 100, Na⁺ form) and the fractions containing theproduct were further purified by reverse phase chromatography(LiChroprep RP-18, CH₃ CN/H₂ O, 5%-50%). The final product was obtainedas a white powder (17 ma, 81%). MS (Electrospray) m/z (M+H)⁺ : calcd1583-1597 (C₅₆ H₇₈ N₁₀ O₂₄ Gd₂, 1590 highest abundance). obsd (the peakof the highest abundance) 1611.4 (M-2H+Na)⁻, 1633.2(M3H+2Na )⁻,804.8(M-3H+Na )²⁻, 793.6(M-2H)²⁻.

The effect of Ca2⁺ on the relaxivity of the complex

In the presence of Ca²⁺, R=5.53 mM⁻¹ sec⁻¹

In the absence of Ca²⁺, R=3.03 mM⁻¹ sec⁻¹

The effect of pH on the relaxivity of the complex

The T1 of the Gd3⁺ -complex (0.4 mM in the buffer containing 100 mM KCl,10 mM MOPS, 2 mM K₂ H₂ EGTA or 2 mM K₂ CaEGTA) was measured underdifferent pH. Changing pH from 6.80 to 7.40 in 0.2 pH unit steps hadminimum effects on the relaxivity of the complex, either in the presenceor in the absence of Ca²⁺.

    ______________________________________                                        pH              6.80   7.00     7.20 7.40                                     ______________________________________                                        T1 (msec, K.sub.2 H.sub.2 EGTA)                                                               600    605      604  608                                        T1 (msec, K.sub.2 CaEGTA) 390 393 394 397                                   ______________________________________                                    

The effect of Mg²⁺ on the relaxivity of the complex

The T1 of the Gd³⁺ -complex (0.4 mM in the buffer containing 132 mM KCl,10 mM MOPS, 1 mM K₂ H₂ EGTA, pH 7.20) was measured. Changing Mg²⁺concentration from 0 to 20 mM had minimum effects on the relaxivity ofthe complex.

    ______________________________________                                        Mg.sup.2+  (mM)                                                                         0      1        2    5      10   20                                   T1 (msec) 607 602 601 609 607 610                                           ______________________________________                                    

We claim:
 1. agent comprising:a) a Gd(III) ion bound to a chelator suchthat said Gd(III) ion has coordination atoms in at least 5 coordinationsites of said Gd(III) ion; b) a blocking moiety covalently attached tosaid chelator which hinders the rapid exchange of water in the remainingcoordination sites;wherein said blocking moiety is capable ofinteracting with a target substance such that the exchange of water inthe remaining coordination sites is increased.
 2. An MRI agent accordingto claim 1 wherein said Gd(III) ion has coordination atoms in at least 6coordination sites of said Gd(III) ion.
 3. An MRI agent according toclaim 1 wherein said Gd(III) ion has coordination atoms in at least 7coordination sites of said Gd(III) ion.
 4. An MRI agent according toclaim 1 wherein said chelator is DOTA or substituted DOTA.
 5. An MRIagent according to claim 1 wherein said chelator is DTPA or substitutedDTPA.
 6. An MRI agent having the formula: ##STR21## wherein M is aparamagnetic metal ion selected from the group consisting of Gd(III),Fe(III), Mn(II), Yt(III), Cr(III) and Dy(III);A, B, C and D are eithersingle bonds or double bonds; X₁, X₂, X₃ and X₄ are --OH, --COO--, --CH₂OH --CH₂ COO--, or a blocking moiety; R₁ -R₁₂ are hydrogen, alkyl, aryl,phosphorus moiety, or a blocking moiety;wherein at least one of X₁ -X₄and R₁ -R₁₂ is a blocking moiety.
 7. An MRI agent according to claim 6wherein at least one of R₉, R₁₀, R₁₁, or R₁₂ is an alkyl group.
 8. AnMRI agent according to claim 6 wherein X₁ is a blocking moiety and R₉ isan alkyl group.
 9. An MRI agent according to claim 6 wherein saidblocking moiety is a peptide.
 10. An MRI agent according to claim 6wherein A, B, C and D are single bonds, R₁ -R₁₂ are hydrogen, and eachR₂₆ is --CH₂ O--.
 11. An MRI agent comprising:a) at least a firstparamagnetic metal ion bound to a first complex, said first complexcomprising:i) a first chelator; and ii) a blocking moiety covalentlyattached to said first chelator which binds in at least a firstcoordination site of said first metal ion and which is capable ofinteracting with a target substance such that the exchange of water inat least said first coordination site of said first metal ion isincreased; and b) at least a second paramagnetic metal ion bound to asecond complex, said second complex comprising:i) a second chelator; andii) a blocking moiety covalently attached to said second chelator whichbinds in at least a first coordination site of said second metal ion andwhich is capable of interacting with a target substance such that theexchange of water in at least said first coordination site of saidsecond metal ion is increased.
 12. An MRI agent according to claim 11wherein said first complex and said second complex are attached via alinker.
 13. An MRI agent according to claim 11 wherein said linker is apolymer.
 14. An MRI agent comprising at least a first MRI duplex moietycomprising:a) a first chelator comprising a first paramagnetic metalion; b) a second chelator comprising a second paramagnetic metal ion; c)a blocking moiety covalently attached to at least one of said first orsaid second chelators, said blocking moiety providing at least a firstcoordination atom of each of said first and said second metal ions andwhich is capable of interacting with a target substance such that theexchange of water in at least a first coordination site in at least oneof said metal ions is increased.
 15. An MRI agent composition accordingto claim 14 further comprising at least a second MRI duplex moiety. 16.An MRI agent composition according to claim 15 wherein said first andsaid second MRI duplexes are attached via a linker.
 17. An MRI agentcomposition according to claim 16 wherein said linker is a polymer. 18.A composition comprising a polymer comprising at least one covalentlylinked MRI contrast agent comprising a paramagnetic metal ion bound to acomplex, said complex comprising:a) a chelator; and b) a blocking moietycovalently attached to said chelator which binds in at least a firstcoordination site of said metal ion and which is capable of interactingwith a target substance such that the exchange of water in at least saidfirst coordination site is increased.
 19. A composition according toclaim 18 wherein said polymer comprises a plurality of said MRI contrastagents.
 20. A composition according to claim 18 wherein said complexfurther comprises a second chelator comprising a second paramagneticmetal ion, and said blocking moiety provides at least a firstcoordination atom for each of said paramagnetic metal ions.
 21. Acomposition according to claim 18 wherein said polymer is a polyaminoacid.
 22. A composition according to claim 21 wherein said polyaminoacid is polylysine.
 23. A composition according to claim 18 wherein saidpolymer has a molecular weight of less than 40 kD.
 24. A compositionaccording to claim 18 wherein said polymer has a molecular weight ofless than 25 kD.
 25. A composition according to claim 18 wherein saidpolymer has a molecular weight of less than 15 kD.
 26. A compositionaccording to claim 18 wherein said polymer has a molecular weight ofless than 10 kD.
 27. An MRI agent according to claim 18 having theformula: ##STR22## wherein M is a paramagnetic metal ion selected fromthe group consisting of Gd(III), Fe(III), Mn(II), Yt(III), Cr(III) andDy(III);A, B, C and D are either single bonds or double bonds; X₁, X₂,X₃ and X₄ are --OH, --COO--, --CH₂ OH or --CH₂ COO--; R₁ -R₁₂ arehydrogen, alkyl, aryl, or a phosphorus moiety; and R₂₆ is a linkermoiety.
 28. An MRI agent according to claim 18 having the formula:##STR23## wherein M is a paramagnetic metal ion selected from the groupconsisting of Gd(III), Fe(III), Mn(II), Yt(III), Cr(III) and Dy(III);A,B, C and D are either single bonds or double bonds; X₁, X₂, X₃ and X₄are --OH, --COO--, --CH₂ OH or --CH₂ COO--; and R₁ -R₁₂ are hydrogen,alkyl, aryl, or a phosphorus moiety.
 29. A method of magnetic resonanceimaging of a cell, tissue or patient comprising administering an MRIagent according to claim 1 to a cell, tissue or patient and rendering amagnetic resonance image of said cell, tissue or patient.
 30. A methodof magnetic resonance imaging of a cell, tissue or patient comprisingadministering an MRI agent according to claim 6 to a cell, tissue orpatient and rendering a magnetic resonance image of said cell, tissue orpatient.
 31. A method of magnetic resonance imaging of a cell, tissue orpatient comprising administering an MRI agent according to claim 11 to acell, tissue or patient and rendering a magnetic resonance image of saidcell, tissue or patient.
 32. A method of magnetic resonance imaging of acell, tissue or patient comprising administering an MRI agent accordingto claim 14 to a cell, tissue or patient and rendering a magneticresonance image of said cell, tissue or patient.
 33. A method ofmagnetic resonance imaging of a cell, tissue or patient comprisingadministering an MRI agent according to claim 18 to a cell, tissue orpatient and rendering a magnetic resonance image of said cell, tissue orpatient.